Mary Helen Barcellos-Hoff

The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression

3D cell cultures are rapidly becoming the method of choice for the physiologically relevant modeling of many aspects of non‐malignant and malignant cell behavior ex vivo. Nevertheless, only a limited number of distinct cell types have been evaluated in this assay to date. Here we report the first large scale comparison of the transcriptional profiles and 3D cell culture phenotypes of a substantial panel of human breast cancer cell lines. Each cell line adopts a colony morphology of one of four main classes in 3D culture. These morphologies reflect, at least in part, the underlying gene expression profile and protein expression patterns of the cell lines, and distinct morphologies were also associated with tumor cell invasiveness and with cell lines originating from metastases. We further demonstrate that consistent differences in genes encoding signal transduction proteins emerge when even tumor cells are cultured in 3D microenvironments.

The Influence of Extracellular Matrix on Gene Expression: Is Structure the Message?

SUMMARY The study of the regulation of gene expression in cultured cells, particularly in epithelial cells, has been both hampered and facilitated by the loss of function that accompanies culture on traditional plastic substrata. Initially, investigations of differentiated function were thwarted by the inadequacy of tissue culture methods developed to support growth of mesenchymal cells. However, with the recognition that the unit of function in higher organisms is larger than the cell itself, and that gene expression is dependent upon cell interactions with hormones, substrata and other cells, came the understanding that the epithelial cell phenotype is profoundly influenced by the extra-cellular environment. In the last decade research on epithelial cells has centred on culture conditions that recreate the appropriate environment for function with very promising and important results. The investigations into the modulation of phenotype in culture produced not only a better model, but also contributed to a better understanding of the regulation of normal function. Using cultured mammary gland epithelial cells as a primary model of these interactions, our studies of gene expression are based on three premises. That the extracellular matrix (ECM) on which the cells sit is an extension of the cells and an active participant in the regulation of cellular function; i.e. the ECM is an ‘informational’ entity in the sense that it receives, imparts and integrates structural and functional signals. That ECM-induced functional differentiation in the mammary gland is mediated through changes in cell shape, i.e. that the structure is in large part ‘the message’ required to maintain differentiated gene expression. That the unit of function includes the cell plus its extracellular matrix; in a larger context, the unit is the organ itself. These tenets and the data presented below are consistent with a model of ‘Dvnamic Reciprocity’, where the ECM is postulated to exert an influence on gene expression via transmembrane proteins and cytoskeletal components. In turn, cytoskeletal association with polyribosomes affects mRNA stability and rates of protein synthesis, while its interaction with the nuclear matrix could affect mRNA processing and, possibly, rates of transcription.

The influence of the microenvironment on the malignant phenotype

Normal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. As tissue becomes cancerous, there are reciprocal interactions between neoplastic cells, adjacent normal cells such as stroma and endothelium, and their microenvironments. The current dominant paradigm wherein multiple genetic lesions provide both the impetus for, and the Achilles heel of, cancer might be inadequate to understand cancer as a disease process. In the following brief review, we will use selected examples to illustrate the influence of the microenvironment in the evolution of the malignant phenotype. We will also discuss recent studies that suggest novel therapeutic interventions might be derived from focusing on microenvironment and tumor cells interactions.

Radiation and the microenvironment – tumorigenesis and therapy

Radiation rapidly and persistently alters the soluble and insoluble components of the tissue microenvironment. This affects the cell phenotype, tissue composition and the physical interactions and signalling between cells. These alterations in the microenvironment can contribute to carcinogenesis and alter the tissue response to anticancer therapy. Examples of these responses and their implications are discussed with a view to therapeutic intervention.

Extracellular signaling through the microenvironment: A hypothesis relating carcinogenesis, bystander effects, and genomic instability

Abstract Barcellos-Hoff, M. H. and Brooks, A. L. Extracellular Signaling through the Microenvironment: A Hypothesis Relating Carcinogenesis, Bystander Effects, and Genomic Instability. Radiat. Res. 156, 618–627 (2001). Cell growth, differentiation and death are directed in large part by extracellular signaling through the interactions of cells with other cells and with the extracellular matrix; these interactions are in turn modulated by cytokines and growth factors, i.e. the microenvironment. Here we discuss the idea that extracellular signaling integrates multicellular damage responses that are important deterrents to the development of cancer through mechanisms that eliminate abnormal cells and inhibit neoplastic behavior. As an example, we discuss the action of transforming growth factor β (TGFB1) as an extracellular sensor of damage. We propose that radiation-induced bystander effects and genomic instability are, respectively, positive and negative manifestations of this homeostatic process. Bystander effects exhibited predominantly after a low-dose or a nonhomogeneous radiation exposure are extracellular signaling pathways that modulate cellular repair and death programs. Persistent disruption of extracellular signaling after exposure to relatively high doses of ionizing radiation may lead to the accumulation of aberrant cells that are genomically unstable. Understanding radiation effects in terms of coordinated multicellular responses that affect decisions regarding the fate of a cell may necessitate re-evaluation of radiation dose and risk concepts and provide avenues for intervention.

Models for evaluating agents intended for the prophylaxis, mitigation and treatment of radiation injuries. Report of an NCI Workshop, December 3-4, 2003

Abstract Stone, H. B., Moulder, J. E., Coleman, C. N., Ang, K. K., Anscher, M. S., Barcellos-Hoff, M. H., Dynan, W. S., Fike, J. R., Grdina, D. J., Greenberger, J. S., Hauer-Jensen, M., Hill, R. P., Kolesnick, R. N., MacVittie, T. J., Marks, C., McBride, W. H., Metting, N., Pellmar, T., Purucker, M., Robbins, M. E., Schiestl, R. H., Seed, T. M., Tomaszewski, J., Travis, E. L., Wallner, P. E., Wolpert, M. and Zaharevitz, D. Models for Evaluating Agents Intended for the Prophylaxis, Mitigation and Treatment of Radiation Injuries. Report of an NCI Workshop, December 3–4, 2003. Radiat. Res. 162, 711–728 (2004). To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.

How Do Tissues Respond to Damage at the Cellular Level? The Role of Cytokines in Irradiated Tissues

The capacity of ionizing radiation to affect tissue function, control tumor growth and elicit pathological sequelae has been attributed in great part to its effects on cellular DNA, which, as the transmitter of genetic information, can both register damage and perpetuate it. Nonetheless, multicellular organisms function as the result of the cooperation of many cell types. What then occurs when individual cells are damaged by ionizing radiation? Is tissue response a sum of cellular effects such as cell death and DNA damage? Or does the tissue respond as a coherent unit to the damage of its parts? In this paper, data in support of the latter model that indicate a role for cytokines, in particular transforming growth factor beta1, as critical components of extracellular signaling pathways that mediate tissue response to radiation will be reviewed. The key to manipulating the consequences of radiation exposure lies in understanding the complex interplay of events initiated at the cellular level, but acting on the tissue.

Conditional Overexpression of Active Transforming Growth Factor β1 In vivo Accelerates Metastases of Transgenic Mammary Tumors

To address the role of transforming growth factor (TGF) β in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-β on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-β1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-β1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-β1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-β and increased metastases. Antisense-mediated inhibition of TGF-β1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-β in hosts with established TGF-β-responsive cancers can rapidly accelerate metastatic progression.

Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species.

Abstract Jobling, M. F., Mott, J. D., Finnegan, M. T., Jurukovski, V., Erickson, A. C., Walian, P. J., Taylor, S. E., Ledbetter, S., Lawrence, C. M., Rifkin, D. B. and Barcellos-Hoff, M. H. Isoform-Specific Activation of Latent Transforming Growth Factor β (LTGF-β) by Reactive Oxygen Species. Radiat. Res. 166, 839–848 (2006). The three mammalian transforming growth factor β (TGF-β) isoforms are each secreted in a latent complex in which TGF-β homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-β from its LAP, called activation, is required for binding of TGF-β to cellular receptors, making extracellular activation a critical regulatory point for TGF-β bioavailability. Our previous work demonstrated that latent TGF-β1 (LTGF-β1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-β1 redox sensitive. First, we compared LTGF-β1, LTGF-β2 and LTGF-β3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-β1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH· availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-β1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-β1 did not alter its ability to associate with LAP, but exposing LAP-β1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-β1 with a mild reducing agent restored its ability to neutralize TGF-β1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-β1 triggers a conformational change that releases TGF-β1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-β1 as critical to ROS-mediated activation. We propose that LTGF-β1 contains a redox switch centered at methionine 253, which allows LTGF-β1 to act uniquely as an extracellular sensor of oxidative stress in tissues.

Latent transforming growth factor-β activation in mammary gland - Regulation by ovarian hormones affects ductal and alveolar proliferation

Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling.