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The role of "aerobic glycolysis" in cancer has been examined often in the past. Results from those studies, most of which were performed on two dimensional conditions (2D, tissue culture plastic), demonstrate that aerobic glycolysis occurs as a consequence of oncogenic events. These oncogenic events often drive malignant cell growth and survival. Although 2D based experiments are useful in elucidating the molecular mechanisms of oncogenesis, they fail to take contributions of the extracellular microenvironment into account. Indeed we, and others, have shown that the cellular microenvironment is essential in regulating processes that induce and/or suppress the malignant phenotype/properties. This regulation between the cell and its microenvironment is both dynamic and reciprocal and involves the integration of cellular signaling networks in the right context. Therefore, given our previous demonstration of the effect of the microenvironment including tissue architecture and media composition on gene expression and the integration of signaling events observed in three-dimension (3D), we hypothesized that glucose uptake and metabolism must also be essential components of the tissue's signal "integration plan" - that is, if uptake and metabolism of glucose were hyperactivated, the canonical oncogenic pathways should also be similarly activated. This hypothesis, if proven true, suggests that direct inhibition of glucose metabolism in cancer cells should either suppress or revert the malignant phenotype in 3D. Here, we review the up-to-date progress that has been made towards understanding the role that glucose metabolism plays in oncogenesis and re-establishing basally polarized acini in malignant human breast cells., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Since the introduction of the cancer stem cell (CSC) hypothesis, accumulating evidence shows that most cancers present stem-like niches. However, therapies aimed at targeting this niche have not been as successful as expected. New evidence regarding CSCs hierarchy, similarities with normal tissue stem cells and cell plasticity might be key in understanding their role in cancer biology and how to efficiently eliminate them. In this Chapter, we discuss what is known in breast and prostate CSCs from their initial discoveries to the current therapeutic efforts in the field. Future challenges towards better CSC identification and isolation strategies will be key to shed light into how CSCs could accurately be targeted in combination to traditional therapies to ultimately prolong patient survival., (© 2019 Elsevier Inc. All rights reserved.)

Intercellular communication is essential for glandular functions and tissue homeostasis. Gap junctions couple cells homotypically and heterotypically and co-ordinate reciprocal responses between the different cell types. Connexins (Cxs) are the main mammalian gap junction proteins, and the distribution of some Cx subtypes in the heterotypic gap junctions is not symmetrical; in the murine mammary gland, Cx26, Cx30 and Cx32 are expressed only in the luminal epithelial cells and Cx43 is expressed only in myoepithelial cells. Expression of all four Cxs peaks during late pregnancy and throughout lactation suggesting essential roles for these proteins in the functional secretory activity of the gland. Transgenic (Tg) mice over-expressing Cx26 driven by keratin 5 promoter had an unexpected mammary phenotype: the mothers were unable to feed their pups to weaning age leading to litter starvation and demise in early to mid-lactation. The mammary gland of K5-Cx26 female mice developed normally and produced normal levels of milk protein, suggesting a defect in delivery rather than milk production. Because the mammary gland of K5-Cx26 mothers contained excessive milk, we hypothesized that the defect may be in an inability to eject the milk. Using ex vivo three-dimensional mammary organoid cultures, we showed that tissues isolated from wild-type FVB females contracted upon treatment with oxytocin, whereas, organoids from Tg mice failed to do so. Unexpectedly, we found that ectopic expression of Cx26 in myoepithelial cells altered the expression of endogenous Cx43 resulting in impaired gap junction communication, demonstrated by defective dye coupling in mammary epithelial cells of Tg mice. Inhibition of gap junction communication or knock-down of Cx43 in organoids from wild-type mice impaired contraction in response to oxytocin, recapitulating the observations from the mammary glands of Tg mice. We conclude that Cx26 acts as a trans-dominant negative for Cx43 function in myoepithelial cells, highlighting the importance of cell type-specific expression of Cxs for optimal contractile function of the mammary myoepithelium., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Intact organ structure is essential in maintaining tissue specificity and cellular differentiation. Small physiological or genetic variations lead to changes in microanatomy that, if persistent, could have functional consequences and may easily be masked by the heterogeneity of tissue anatomy. Current imaging techniques rely on histological, two-dimensional sections requiring sample manipulation that are essentially two dimensional. We have developed a method for three-dimensional imaging of whole-mount, unsectioned mammalian tissues to elucidate subtle and detailed micro- and macroanatomies in adult organs and embryos. We analyzed intact or dissected organ whole mounts with laser scanning-based tissue autofluorescence/fluorescence imaging (LS-TAFI). We obtained clear visualization of microstructures within murine mammary glands and mammary tumors and other organs without the use of immunostaining and without probes or fluorescent reporter genes. Combining autofluorescence with reflected light signals from chromophore-stained tissues allowed identification of individual cells within three-dimensional structures of whole-mounted organs. This technique could be useful for rapid diagnosis of human clinical samples and possibly the effect of subtle variations such as low dose radiation., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

This Commentary highlights two articles in this issue of the American Journal of Pathology, discussing the implications of stromal expression of caveolin-1 in breast cancer.

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.

Transforming growth factor-alpha (TGFalpha) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK(ERK1,2)); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK(ERK1,2) for 1 h, induced by TGFalpha, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK(ERK1,2), induced by FGF7, led to growth without branching. Unlike TGFalpha, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGFalpha indicated that the FGF7-induced MAPK(ERK1,2) signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGFalpha-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK(ERK1,2) activation.

The importance of microenvironment and context in regulation of tissue-specific genes is well established. DNA exposure to or the sequestration from nucleases detects differences in higher order chromatin structure in intact cells without disturbing cellular or tissue architecture. To investigate the relationship between chromatin organization and tumor phenotype, we used an established three-dimensional assay in which normal and malignant human breast cells can be easily distinguished by the morphology of the structures they make (acinus-like versus tumor-like, respectively). We show that these phenotypes can be distinguished also by sensitivity to AluI digestion in which the malignant cells resist digestion relative to nonmalignant cells. Treatment of T4-2 breast cancer cells in three-dimensional culture with cAMP analogs or a phosphatidylinositol 3-kinase inhibitor not only reverted their phenotype from nonpolar to polar acinar-like structures but also enhanced chromatin sensitivity to AluI. By using different cAMP analogs, we show that cAMP-induced phenotypic reversion, polarization, and shift in DNA organization act through a cAMP-dependent protein-kinase A-coupled signaling pathway. Importantly, inhibitory antibody to fibronectin produced the same effect. These experiments underscore the concept that modifying the tumor microenvironment can alter the organization of tumor cells and demonstrate that architecture and global chromatin organization are coupled and highly plastic.

Almost three decades ago, we presented a model where the extracellular matrix (ECM) was postulated to influence gene expression and tissue-specificity through the action of ECM receptors and the cytoskeleton. This hypothesis implied that ECM molecules could signal to the nucleus and that the unit of function in higher organisms was not the cell alone, but the cell plus its microenvironment. We now know that ECM invokes changes in tissue and organ architecture and that tissue, cell, nuclear, and chromatin structure are changed profoundly as a result of and during malignant progression. Whereas some evidence has been generated for a link between ECM-induced alterations in tissue architecture and changes in both nuclear and chromatin organization, the manner by which these changes actively induce or repress gene expression in normal and malignant cells is a topic in need of further attention. Here, we will discuss some key findings that may provide insights into mechanisms through which ECM could influence gene transcription and how tumor cells acquire the ability to overcome these levels of control.

Increased expression of matrix metalloproteinases (MMPs) is associated with nearly every tumor type. Although many studies have shown that MMPs can promote malignancy, recent evidence has revealed that MMPs can play a causative role also in the earliest stages of cancer development. A complex story is now emerging in which MMPs not only compromise cell-cell and cell-substratum adhesion processes that impact genomic surveillance mechanisms but also act directly on molecules at the cell surface to stimulate physiological processes that cause genetic alterations. Delineating the mechanisms involved in these processes and identifying how they are coordinated in vivo could aid identification of the crucial contribution of MMPs to tumorigenesis.

Given that expression of many genes changes when cells become malignant or are placed in different microenvironments, we asked whether these changes were accompanied by global reorganization of chromatin. We reasoned that sequestration or exposure of chromatin-sensitive sites to restriction enzymes could be used to detect this reorganization. We found that AluI-sensitive sites of nonmalignant cells were relatively more exposed compared to their malignant counterparts in cultured cells and human tumor samples. Changes in exposure and sequestration of AluI-sensitive sites in normal fibroblasts versus fibrosarcoma or those transfected with oncogenes, nonmalignant breast cells versus carcinomas and poorly metastatic versus highly invasive melanoma were shown to be independent of the cell cycle and may be influenced by proteins rich in disulfide bonds. Remarkably, regardless of degree of malignancy, AluI-sensitive sites became profoundly sequestered when cells were incubated with laminin, Matrigel, or a circular RGD peptide (RGD-C), but became exposed when cells were placed on collagen I or in serum-containing medium. Disruption of the actin cytoskeleton led to exposure, whereas disruption of microtubules or intermediate filaments exerted a sequestering effect. Thus, AluI-sensitive sites are more sequestered with increasing malignant behavior, but the sequestration and exposure of these sites is exquisitely sensitive to information conferred to the cell by molecules and biomechanical forces that regulate cellular and tissue architecture.

A breast carcinoma biopsy showed cytochemical evidence of epithelial mesenchymal transition and an alpha-smooth muscle actin-positive stromal reaction. To study the lineage, and the nature of the cells in the stromal reaction, we derived a novel cell line, HBFL-1, from the explanted biopsy. HBFL-1 cells are immortal and exhibit a shared non-random X-chromosome inactivation pattern with the epithelial tumor of origin. Yet they closely resemble normal, finite-life-span fibroblasts by morphology, lack of tumor formation in nude mice, marker expression profile, protein pattern using two-dimensional gel electrophoresis and the ability to undergo myofibroblast conversion. HBFL-1 interacts reciprocally with tumor cells in collagen gel to induce activation of MMP2, leading to tumor-like behavior of epithelial colonies. In vivo, HBFL-1 cells resembled normal-derived myofibroblasts and conferred a significant 3.5- to 7-fold increase in MCF-7 tumor size in nude mice. However, that they were indeed not normal fibroblasts was revealed by residual keratin expression and formation of epithelial microfoci in a reconstituted basement membrane and in nude mice. We conclude that breast cancer can generate its own nonmalignant stroma and that one function for this is that of a reciprocal interaction with epithelial tumor cells to facilitate tumor growth.

The current paradigm for cancer initiation and progression rests on the groundbreaking discoveries of oncogenes and tumor suppressor genes. This framework has revealed much about the role of genetic alterations in the underlying signaling pathways central to normal cellular function and to tumor progression. However, it is clear that single gene theories or even sequential acquisition of mutations underestimate the nature of the genetic and epigenetic changes in tumors, and do not account for the observation that many cancer susceptibility genes (e.g. BRCA1, APC) show a high degree of tissue specificity in their association with neoplastic transformation. Therefore, the cellular and tissue context itself must confer additional and crucial information necessary for mutated genes to exert their influence. A considerable body of evidence now shows that cell-cell and cell-extracellular matrix (ECM) interactions are essential organizing principles that help define the nature of the tissue context, and play a crucial role in regulating homeostasis and tissue specificity. How this context determines functional integrity, and how its loss can lead to malignancy, appears to have much to do with tissue structure and polarity.

The matrix metalloproteinase stromelysin-1 plays a central role during mammary gland development and tumor progression. To gain insight into the regulation of stromelysin-1 gene expression, the murine stromelysin-1 promoter was cloned and transfected into mouse mammary epithelial cells displaying various degrees of malignancy. A reconstituted basement membrane inhibited stromelysin-1 promoter activity in functionally normal cells, had little effect on moderately malignant cells and up-regulated the promoter in highly malignant cells. Spreading of normal and malignant cells was reduced by a reconstituted basement membrane, compared to a plastic substratum. Preventing spreading by maintenance of cells in suspension culture, regulated stromelysin-1 promoter activity in a manner similar to that on a reconstituted basement membrane. Conversely, increasing spreading by augmenting substratum adhesivity up-regulated stromelysin-1 promoter activity in tumor cells. In cells with reduced spreading in the presence of reconstituted basement membrane and in suspension culture, actin stress fibers were replaced by cortical actin bundles. In tumor cells, but not in functionally normal cells, treatment with phorbol diesters also resulted in accumulation of cortical actin and increased stromelysin-1 promoter activity. Consistent with an epithelial-to-mesenchymal conversion, regulation of stromelysin-1 gene expression in highly malignant cells was similar to its regulation in mammary fibroblasts. We conclude that the switch in transcriptional regulation of stromelysin-1 expression that occurs during epithelial-to-mesenchymal transition and conversion to tumorigenicity is related to altered regulation of signals from the cytoarchitecture.

The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and alpha-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around., (Copyright 1999 Academic Press.)

A growing number of studies have established reciprocal linkages between extracellular matrix (ECM)-integrins, growth factor signaling and cell-cell adhesion molecules. ECM-dependent tissue-specific gene expression has also been linked to chromatin remodeling. With respect to tissue morphogenesis and differentiation, crosstalk has been established between the ECM and the homeobox morphoregulatory genes. Each of these linkages is profoundly influenced by the cell's microenvironment and the resulting tissue form. Thus for a cell to achieve a differentiated phenotype, the ECM molecules and their receptors must integrate both form and function. In contrast, mutated genes and aberrant interactions with the microenvironment conspire to undermine this integration, often resulting in malignant transformation.

Extracellular matrix and extracellular matrix-degrading matrix metalloproteinases play a key role in interactions between the epithelium and the mesenchyme during mammary gland development and disease. In patients with breast cancer, the mammary mesenchyme undergoes a stromal reaction, the etiology of which is unknown. We previously showed that targeting of an autoactivating mutant of the matrix metalloproteinase stromelysin-1 to mammary epithelia of transgenic mice resulted in reduced mammary function during pregnancy and development of preneoplastic and neoplastic lesions. Here we examine the cascade of alterations before breast tumor formation in the mammary gland stroma once the expression of the stromelysin-1 transgene commences. Beginning in postpubertal virgin animals, low levels of transgene expression in mammary epithelia led to increased expression of endogenous stromelysin-1 in stromal fibroblasts and up-regulation of other matrix metalloproteinases, without basement membrane disruption. These changes were accompanied by the progressive development of a compensatory reactive stroma, characterized by increased collagen content and vascularization in glands from virgin mice. This remodeling of the gland affected epithelial-mesenchymal communication as indicated by inappropriate expression of tenascin-C starting by day 6 of pregnancy. This, together with increased transgene expression, led to basement membrane disruption starting by day 15 of pregnancy. We propose that the highly reactive stroma provides a prelude to breast epithelial tumors observed in these animals.

In this brief review, the development of breast cancer is discussed from the vantage of phenotypic differentiation, similar to what has been considered over the years for leukemias and melanomas, both of which express easily visible differentiation markers (Hart and Easty, 1991; Clarke et al., 1995; Lynch, 1995; Sachs, 1996; Sledge, 1996). The review is divided into a theoretical background for human breast differentiation and a discussion of recent experimental results in our laboratories with differentiation of breast epithelial cells. In the theoretical background, in situ markers of differentiation of normal breast and carcinomas are discussed with emphasis on their possible implications for tumor therapy. So far, most of the emphasis regarding differentiation therapy of tumors has been focused on the possible action of soluble factors, such as colony-stimulating factors in leukemias and retinoic acids in solid tumors (Lotan, 1996; Sachs, 1996). However, an emerging and promising new avenue in this area appears to point to additional factors, such as the cellular form and extracellular matrix (ECM) (Bissel et al., 1982; Bissel and Barcellos-Hoff, 1987; Ingber, 1992). The recent interest in these parameters has evolved along with an increasing understanding of the molecular composition of the ECM, and of the molecular basis of the classical findings that normal cell--in contrast to tumor cells--are anchorage dependent for survival and growth (Folkman and Moscona, 1978; Hannigan et al., 1996). We now know that this is the case for epithelial as well as fibroblastic cells, and that interaction with ECM is crucial for such regulation. Indeed, ECM and integrins are emerging as the central regulators of differentiation, apoptosis, and cancer (Boudreau et al., 1995; Boudreau and Bissel, 1996; Werb et al., 1996; Bissell, 1997; Weaver, et al., 1997). In the experimental part, we elaborate on our own recent experiments with functional culture models of the human breast, with particular emphasis on how "normal" and cancer cells could be defined within a reconstituted ECM. Special attention is given to integrins, the prominent ECM receptors. We further discuss a number of recent experimental results, all of which point to the same conclusion: namely that phenotypic reversion toward a more normal state for epithelial tumors is no longer an elusive goal. Thus "therapy by differentiation" could be broadened to include not only blood-borne tumors, but also solid tumors of epithelial origin.

A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution.

The basis for specific changes in differentiation that accompany ras-oncogene-mediated transformation are not understood. When short-term cultures of fibroblast-like cells from adult rat adrenal glands were transformed with Kirsten murine sarcoma virus (KiMSV), the original stromal characteristics of the target cells (metachromatic extracellular matrix, high collagen production, collagen incorporation into pericellular matrix and a fibroblastic morphology and growth pattern) diminished. In contrast, parenchymal differentiation markers (neutral lipid, delta 5, 3 beta-hydroxysteroid dehydrogenase, 21-hydroxylase and epithelial morphology) were enhanced. These changes in differentiation were initiated concurrently with the over-expression of the transforming protein v-p21, but were unrelated to the levels of v-p21 expression. They were independent of the immortalizing component of transformation mediated by v-K-ras, because they did not take place in spontaneously immortalized lines derived from the same target cells, unless the lines were also transformed with KiMSV. In normal embryonic development, stromal and parenchymal adrenocortical cells arise by divergent differentiation pathways from a common, multipotential mesenchymal precursor. The transformation-induced modulation from a predominantly stromal to a more-parenchymal phenotype is thus reminiscent of reversion to a more primitive, bipotential developmental stage.