Intact Human Breast Research Articles

PRECISE: Preoperative Radiation Therapy to Elicit Critical Immune Stimulating Effects – A Phase 2 Clinical Trial

PurposeRadiotherapy is an under-investigated tool for priming the immune system in intact human breast cancers. We sought here to investigate if a preoperative radiotherapy boost delivered was associated with a significant change in tumor infiltrating lymphocytes in the tumor in estrogen receptor positive, HER2Neu non-amplified breast cancers. Methods and Materials20 patients were enrolled in a phase II clinical trial and received either 7.5Gy x 1 fraction or 2Gy x 5 fractions, completed 6-8 days prior to surgery. Percent stromal tumor infiltrating lymphocytes (TIL) were evaluated on hematoxylin and eosin-stained samples. Short-term safety was assessed based on time to surgery, toxicities, and cosmesis up to 6 months following boost. ResultsStromal TIL increased 6-8 days following completion of boost radiotherapy (median 3.0 (IQR 1.0-6.5) prior to radiotherapy vs. median 5.0 (IQR 1.5-8.0) post radiotherapy, p=0.0037. Zero grade ≥ 3 toxicities up to 6 months following boost were experienced. 94% (16/17) patients with 6 month follow-up cosmetic assessment following breast conservation had good-excellent cosmesis by physician assessment. ConclusionIn this phase II trial, preoperative radiotherapy boost resulted in a short-term increase in stromal TIL with minimal toxicities. Preoperative breast radiotherapy appears to be safe and may be a feasible means for priming the tumor microenvironment.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2015-1158

Marinopyrrole derivatives with sulfide spacers as selective disruptors of Mcl-1 binding to pro-apoptotic protein Bim.

A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.

Identification of novel ROCK inhibitors with anti-migratory and anti-invasive activities

ROCK1 and ROCK2 mediate important processes such as cell migration, invasion and metastasis; making them good targets for the development of antitumor agents. Recently, using a fragment-based approach and X-ray crystallography, we reported on the design and synthesis of novel Rho-kinase inhibitors (RKIs). Here, we selected a pair of RKIs, the closely-related structural analogues RKI-18 (potent; IC50 values of 397 nM (ROCK1) and 349 nM (ROCK2)) and RKI-11 (weak/inactive; IC50 values of 38 µM (ROCK1) and 45 µM (ROCK2), as chemical probes and determined their effects on cytoskeleton organization, signaling, apoptosis, anchorage-dependent and –independent growth, migration and invasion. RKI-18 but not RKI-11 suppresses potently the phosphorylation of the ROCK substrate MLC2 in intact human breast, lung, colon and prostate cancer cells. Furthermore, RKI-18 is highly selective at decreasing the levels of P-MLC2 over those of P-Akt, P-S6 and P-Erk ½. RKI-18 suppresses ROCK-mediated actin fiber formation following stimulation with LPA as well as PAK-mediated lamelipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that the active ROCK inhibitor RKI-18 but not the inactive closely related structural analogue RKI-11 is effective at suppressing malignant transformation suggests that inhibition of ROCK with RKI-18 results in preventing migration, invasion and anchorage-independent growth. The potential of this class of RKIs as anti tumor agents warrants further advanced preclinical studies.

Targeting of Rac GTPases blocks the spread of intact human breast cancer

High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a 3-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using 3-dimensional primary tumour tissue explant cultures.

Fragment-Based and Structure-Guided Discovery and Optimization of Rho Kinase Inhibitors

Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC(50) = 650 nM) and ROCK2 (IC(50) = 670 nM), whereas compound 24 was more selective for ROCK2 (IC(50) = 100 nM) over ROCK1 (IC(50) = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.

Risk of breast cancer with progestins: critical assessment of current data

Whether progestins protect against the risk of breast cancer or enhance that risk has been a major area of controversy over the past several years. Observational studies have reported conflicting results and experimental studies examining whether progestins exert mitogenic or anti-mitogenic actions on breast tissue report divergent results. Based upon a wide range of animal, epidemiologic and clinical data, most investigators agree that estrogens contribute to the development of breast neoplasms. However, the additional effect of progestins on this risk has been the subject of substantial discussion and controversy. A variety of experiments have been carried out using human breast cancer cells grown in vitro and as xenografts in nude mice. These studies demonstrated both mitogenic and anti-mitogenic effects depending upon the precise experimental conditions. Two potential reasons for these differences include differential metabolism of progestins into inhibitory pregnenes or stimulatory 5-alpha-reduced pregnanes or the presence of a protein (GPR 30) which allows the anti-mitogenic effects of progestins to be manifest. Based upon the conflicting nature of the results in experimental studies, we believe that only data in patients provide substantial insight into the actions of progestins on the intact human breast. Studies have now demonstrated that cell proliferation and breast density is higher during the luteal than during the follicular phase of the menstrual cycle. In postmenopausal women, long-term exposure to estrogen plus a progestin results in a marked enhancement of proliferation of the terminal duct lobular units as well as in breast density. These data, taken together, provide substantial evidence that progestins are mitogenic on the human breast when given long term to postmenopausal women. To critically evaluate the observational studies regarding breast cancer risk from progestins, we developed a set of stringent criteria for acceptance of individual studies. Four of the five studies meeting these criteria reported a greater risk of breast cancer with combination estrogen/progestin regimens than with estrogen alone. More importantly, the first randomized, prospective, controlled trial of the risk of breast cancer with an estrogen/progestin combination (the Women’s Health Initiative Study) has now been published. This study reported a 26% increased relative risk of breast cancer with the estrogen/progestin combination. Based upon these data, we believe that progestins do add to the risk of breast cancer over and above that imparted by estrogen alone. The attributable risk during use for 5 years or less is small but increases logarithmically during long-term use. The majority of data regarding progestins are derived from regimens using MPA. However, we conclude from our analysis that the burden of proof regarding progestins has now shifted. One must now prove that an estrogen/progestin combination is safe with respect to breast cancer rather than having to prove it harmful.

Histomorphologically intact primary human breast lesions and cancers can be propagated in nude mice

The natural history of various human breast lesions and cancer in patient is difficult to study since diagnosis is established only after surgical ablation and therefore the clinical course of the undisturbed lesion in vivo can no longer be followed. Development of an experimental system which can propagate and maintain human breast lesions and cancer, similar to those seen in surgical breast specimens, may be a first step in the analyses of these various entities. We report that histomorphologically intact human breast lesions and cancer can be recreated in an experimental system using athymic nude mice. When the dissociated cells from surgical breast cancer specimens are embedded in extracellular matrices (collagen gel, Matrigel, or a mixture of the two) and then transplanted into athymic nude mice, the transplanted cells undergo morphogenesis to reflect their original phenotype. A mixture of the two matrices most closely mirrored the breast tissue in situ when stained with Alcian blue-PAS which stains for mucins. In this mixture, not only is the histomorphology recreated, but well-established clinical molecular markers including the estrogen receptor, c-erbB-2 (HER-2/neu), and aberrant cell proliferation are maintained. With the use of an appropriate extracellular matrices, surgical specimens of human breast lesions and cancer can be analyzed in an in vivo experimental system.

Estrogen mitogenic action. III. is phenol red a "red herring"?

The reported estrogenic action of phenol red and/or its lipophilic contaminants has led to the widespread use of indicator-free culture medium to conduct endocrine studies in vitro. Because we have recently developed methods to measure large-magnitude estrogen effects in the tissue culture medium containing phenol red, we concluded that the indicator issue required further evaluation. To do this, we selected nine estrogen receptor positive (ER+) cell lines representing four target tissues and three species. We investigated phenol red using five different experimental protocols. First, 17beta-estradiol (E2) responsive growth of all nine ER+ cells lines was compared in the medium with and without the indicator. Second, using representative lines we asked if phenol red was mitogenic in the indicator-free medium. The dose-response effects of phenol red were compared directly to those of E2. Third, we asked if tamoxifen-inhibited growth equally in phenol red-containing and indicator-free medium. This study was based on a report indicating that antiestrogen effects should be seen only in phenol red-containing medium. Fourth, we asked if phenol red displaced the binding of 3H-E2 using ERK intact human breast cancer cells. Fifth, we compared E2 and phenol red as inducers of the progesterone receptor using a human breast cancer cell line. All the experiments presented in this report support the conclusion that the concentration of phenol red contaminants in a standard culture medium available today is not sufficient to cause estrogenic effects. In brief, our studies indicate that the real issue of how to demonstrate estrogenic effects in culture resides elsewhere than phenol red. We have found that the demonstration of sex steroid hormone-mitogenic effects in culture depends upon conditions that maximize the effects of a serum-borne inhibitor(s). When the effects of the inhibitor are optimized, the presence or absence of phenol red makes no everyday difference to the demonstration of estrogen mitogenic effects with several target cell types from diverse species.

Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells

The total concentration of 14 polycyclic aromatic hydrocarbons (PAHs) was determined to be 3400-fold greater in a sediment sample from an industrial site on the St. Lawrence River (SLR), NY, than in a sediment sample from a non-industrial site on the Kinderhook Creek (KC), NY. PAH fractions from extracts of the two environmental samples and two reconstituted mixtures as well as the 14 individual PAHs were examined for their toxic, estrogenic, and antiestrogenic activities using MCF-7 focus, recombinant human estrogen receptor (ER) binding, whole-cell ER binding, and 17β-estradiol (E 2) metabolism assays. PAH fractions from the KC and SLR were antiestrogenic; they significantly inhibited the formation of foci elicited in MCF-7 breast cancer cells by 1 nM E 2. Eight of the 14 individual PAHs, and the reconstituted mixtures were also antiestrogenic. Results from the whole-cell ER binding assay and the radiometric analysis of E 2 metabolism indicate that the PAHs detected in the KC and the SLR environmental samples induce antiestrogenic responses in metabolically intact human breast cancer cells through at least two mechanisms: one involving competition for the ER by a PAH metabolite and the other involving depletion of E 2 through induction of metabolism.

Telomerase Is Controlled by Protein Kinase Cα in Human Breast Cancer Cells

Telomerase, a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in human somatic tissues and becomes activated during tumor progression in most human cancers. To date, little is known about how telomerase is activated and controlled in cancer, although activation is thought to be involved in cancer cell immortalization. Here, we report that human telomerase-associated protein 1 (hTEP1) and the telomerase catalytic subunit (human telomerase reverse transcriptase (hTERT)) are phosphoproteins and that their phosphorylation is a prerequisite for the activation of telomerase in intact human breast cancer cells. Identified by hTEP1 peptide affinity chromatography, protein kinase Calpha mediates the phosphorylation of hTEP1 and hTERT and induces a marked increase in telomerase activity. Thus, phosphorylation of hTEP1 and hTERT by protein kinase Calpha represents an essential step in the generation of a functional telomerase complex in the initiation and maintenance of telomerase activity in human cancer.

Estrogen and progestin bioactivity of foods, herbs, and spices.

In this study we report on the content and bioactivity of plant (phyto) estrogens and progestins in various foods, herbs, and spices, before and after human consumption. Over 150 herbs traditionally used by herbalists for treating a variety of health problems were extracted and tested for their relative capacity to compete with estradiol and progesterone binding to intracellular receptors for progesterone (PR) and estradiol (ER) in intact human breast cancer cells. The six highest ER-binding herbs that are commonly consumed were soy, licorice, red clover, thyme, tumeric, hops, and verbena. The six highest PR-binding herbs and spices commonly consumed were oregano, verbena, tumeric, thyme, red clover and damiana. Some of the herbs and spices found to contain high phytoestrogens and phytoprogestins were further tested for bioactivity based on their ability to regulate cell growth rate in ER (+) and ER (-) breast cancer cell lines and to induce or inhibit the synthesis of alkaline phosphatase, an end product of progesterone action, in PR (+) cells. In general, we found that ER-binding herbal extracts were agonists, much like estradiol, whereas PR-binding extracts, were neutral or antagonists. The bioavailability of phytoestrogens and phytoprogestins in vivo were studied by quantitating the ER-binding and PR-binding capacity of saliva following consumption of soy milk, exogenous progesterone, medroxyprogesterone acetate, or wild mexican yam products containing diosgenin. Soy milk caused a dramatic increase in saliva ER-binding components without a concomitant rise in estradiol. Consumption of PR-binding herbs increased the progestin activity of saliva, but there were marked differences in bioactivity. In summary, we have demonstrated that many of the commonly consumed foods, herbs, and spices contain phytoestrogens and phytoprogestins that act as agonists and antagonists in vivo.

Human breast cancer cells contain a phosphoramidon-sensitive metalloproteinase which can process exogenous big endothelin-1 to endothelin-1: a proposed mitogen for human breast fibroblasts

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Breast lesion discrimination using statistical analysis and shape measures on magnetic resonance imagery

Magnetic resonance images of intact human breast tissue are evaluated using statistical measures and shape analysis. In this paper, the Mahalanobis distance measurement and a related F-statistical value demonstrate that breast lesions are statistically separable from normal breast tissue. The minimum set of parameters to provide first order statistical separability between fibroadenomas, cysts, and carcinomas are TI-weighted, T2-weighted, and Dixon opposed pulse sequences. Tumor shape is quantified by development of a compactness measure and a spatial frequency analysis of the lesion boundary. Malignant lesions are shown to be separable from benign lesions based on quantitative shape measures.

Radiation inactivation of estrogen receptor in intact human breast cancer cells (MCF-7) Lack of energy transfer to the estradiol binding domain

Whole MCF-7 human breast-cancer cells were irradiated at −78°C in a calibrated Gammacel 6OCo irradiator. Freezing or storing conditions induce neither an alteration of the viability of cells nor a change in estradiol binding activity. Hexosaminidase was used as internal marker, and we measured the radiation inactivation size (RIS) of the estrogen receptor in whole cells. After various cell treatments, the estradiol binding unit always presents a molecular mass of 25 kDa. This value, which corresponds to the size of the defined hormone binding domain of the estrogen receptor, suggests that the energy delivered to the protein by the radiation is efficient to inactivate estradiol binding only when the hit occurs directly in the smaller hormone binding domein.