Persistent Phenotypic Responses of Human Mammary Epithelial Cells Induced by Ionizing Radiation

Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. We have shown that irradiating human mammary epithelial cells (HMEC) cultured with that transforming growth factor β1 (TGFβ) can generate a persistent phenotype in daughter cells characterized by spindle cell morphology, increased mesenchymal markers, decreased epithelial markers and increased cellular motility and invasion, which are hallmarks of epithelial to mesenchymal transition (EMT). Neither radiation nor TGFβ alone elicited EMT, although IR increased chronic TGFβ signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/MAPK signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFβ-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFβ, and inhibition of Erk activation, blocked the EMT phenotype. Preliminary studies suggest that eqi-toxic doses of sparsely and densely ionizing radiation resulted in comparable EMT when cells were cultivated in the presence of TGFβ, Furthermore radiation dose response studies show that this effect has a very low threshold in that a single exposure of 3-200 cGy radiation elicits the ‘same’ phenotypic switch, which is consistent with non-targeted effects. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to radiation carcinogenesis

Multidimensional profiling of cell surface proteins and nuclear markers

Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables

Transforming growth factor-β in breast cancer: too much, too late

The contribution of transforming growth factor (TGF)β to breast cancer has been studied from a myriad perspectives since seminal studies more than two decades ago. Although the action of TGFβ as a canonical tumor suppressor in breast is without a doubt, there is compelling evidence that TGFβ is frequently subverted in a malignant plexus that drives breast cancer. New knowledge that TGFβ regulates the DNA damage response, which underlies cancer therapy, reveals another facet of TGFβ biology that impedes cancer control. Too much TGFβ, too late in cancer progression is the fundamental motivation for pharmaceutical inhibition

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

BACKGROUND: Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. METHODS: The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. RESULTS: Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. CONCLUSION: These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis

Promotion of variant human mammary epithelial cell outgrowth by ionizing radiation: an agent-based model supported by in vitro studies

IntroductionMost human mammary epithelial cells (HMEC) cultured from histologically normal breast tissues enter a senescent state termed stasis after 5 to 20 population doublings. These senescent cells display increased size, contain senescence associated beta-galactosidase activity, and express cyclin-dependent kinase inhibitor, p16INK4A (CDKN2A; p16). However, HMEC grown in a serum-free medium, spontaneously yield, at low frequency, variant (v) HMEC that are capable of long-term growth and are susceptible to genomic instability. We investigated whether ionizing radiation, which increases breast cancer risk in women, affects the rate of vHMEC outgrowth.MethodsPre-stasis HMEC cultures were exposed to 5 to 200 cGy of sparsely (X- or gamma-rays) or densely (1 GeV/amu 56Fe) ionizing radiation. Proliferation (bromodeoxyuridine incorporation), senescence (senescence-associated beta-galactosidase activity), and p16 expression were assayed in subcultured irradiated or unirradiated populations four to six weeks following radiation exposure, when patches of vHMEC became apparent. Long-term growth potential and p16 promoter methylation in subsequent passages were also monitored. Agent-based modeling, incorporating a simple set of rules and underlying assumptions, was used to simulate vHMEC outgrowth and evaluate mechanistic hypotheses.ResultsCultures derived from irradiated cells contained significantly more vHMEC, lacking senescence associated beta-galactosidase or p16 expression, than cultures derived from unirradiated cells. As expected, post-stasis vHMEC cultures derived from both unirradiated and irradiated cells exhibited more extensive methylation of the p16 gene than pre-stasis HMEC cultures. However, the extent of methylation of individual CpG sites in vHMEC samples did not correlate with passage number or treatment. Exposure to sparsely or densely ionizing radiation elicited similar increases in the numbers of vHMEC compared to unirradiated controls. Agent-based modeling indicated that radiation-induced premature senescence of normal HMEC most likely accelerated vHMEC outgrowth through alleviation of spatial constraints. Subsequent experiments using defined co-cultures of vHMEC and senescent cells supported this mechanism.ConclusionsOur studies indicate that ionizing radiation can promote the outgrowth of epigenetically altered cells with pre-malignant potential

The TGF-β/Smad Repressor TG-Interacting Factor 1 (TGIF1) Plays a Role in Radiation-Induced Intestinal Injury Independently of a Smad Signaling Pathway

Despite advances in radiation delivery protocols, exposure of normal tissues during the course of radiation therapy remains a limiting factor of cancer treatment. If the canonical TGF-β/Smad pathway has been extensively studied and implicated in the development of radiation damage in various organs, the precise modalities of its activation following radiation exposure remain elusive. In the present study, we hypothesized that TGF-β1 signaling and target genes expression may depend on radiation-induced modifications in Smad transcriptional co-repressors/inhibitors expressions (TGIF1, SnoN, Ski and Smad7). In endothelial cells (HUVECs) and in a model of experimental radiation enteropathy in mice, radiation exposure increases expression of TGF-β/Smad pathway and of its target gene PAI-1, together with the overexpression of Smad co-repressor TGIF1. In mice, TGIF1 deficiency is not associated with changes in the expression of radiation-induced TGF-β pathway-related transcripts following localized small intestinal irradiation. In HUVECs, TGIF1 overexpression or silencing has no influence either on the radiation-induced Smad activation or the Smad3-dependent PAI-1 overexpression. However, TGIF1 genetic deficiency sensitizes mice to radiation-induced intestinal damage after total body or localized small intestinal radiation exposure, demonstrating that TGIF1 plays a role in radiation-induced intestinal injury. In conclusion, the TGF-β/Smad co-repressor TGIF1 plays a role in radiation-induced normal tissue damage by a Smad-independent mechanism

Adipocyte extracellular matrix composition, dynamics and role in obesity

The central role of the adipose tissue in lipid metabolism places specific demands on the cell structure of adipocytes. The protein composition and dynamics of the extracellular matrix (ECM) is of crucial importance for the functioning of those cells. Adipogenesis is a bi-phasic process in which the ECM develops from a fibrillar to a laminar structure as cells move from the commitment phase to the growth phase characterized by storage of vast amounts of triglycerides. Mature adipocytes appear to spend a lot of energy on the maintenance of the ECM. ECM remodeling is mediated by a balanced complement of constructive and destructive enzymes together with their enhancers and inhibitors. ECM remodeling is an energy costing process regulated by insulin, by the energy metabolism, and by mechanical forces. In the obese, overgrowth of adipocytes may lead to instability of the ECM, possibly mediated by hypoxia

Heavy Ion Carcinogenesis and Human Space Exploration

Prior to the human exploration of Mars or long duration stays on the Earth s moon, the risk of cancer and other diseases from space radiation must be accurately estimated and mitigated. Space radiation, comprised of energetic protons and heavy nuclei, has been show to produce distinct biological damage compared to radiation on Earth, leading to large uncertainties in the projection of cancer and other health risks, while obscuring evaluation of the effectiveness of possible countermeasures. Here, we describe how research in cancer radiobiology can support human missions to Mars and other planets

Matrix Metalloproteinase-Induced Epithelial-Mesenchymal Transition in Breast Cancer

Matrix metalloproteinases (MMPs) degrade and modify the extracellular matrix (ECM) as well as cell-ECM and cell-cell contacts, facilitating detachment of epithelial cells from the surrounding tissue. MMPs play key functions in embryonic development and mammary gland branching morphogenesis, but they are also upregulated in breast cancer, where they stimulate tumorigenesis, cancer cell invasion and metastasis. MMPs have been investigated as potential targets for cancer therapy, but clinical trials using broad-spectrum MMP inhibitors yielded disappointing results, due in part to lack of specificity toward individual MMPs and specific stages of tumor development. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells take on the characteristics of invasive mesenchymal cells, and activation of EMT has been implicated in tumor progression. Recent findings have implicated MMPs as promoters and mediators of developmental and pathogenic EMT processes in the breast. In this review, we will summarize recent studies showing how MMPs activate EMT in mammary gland development and in breast cancer, and how MMPs mediate breast cancer cell motility, invasion, and EMT-driven breast cancer progression. We also suggest approaches to inhibit these MMP-mediated malignant processes for therapeutic benefit