Reproductive state of rat mammary gland stroma modulates human breast cancer cell migration and invasion.

It has been established that the invasive behavior of cancer cells can be regulated by alterations in their extracellular environment. We investigated whether extracellular matrix isolated from nulliparous and postlactating (involuting) rat mammary glands differentially modulated the metastatic behavior of human breast cancer cells. Using modified Boyden chamber and three-dimensional culture assays, nulliparous mammary matrix was found to suppress motility and invasion in highly metastatic MDA-MB-435 cells, whereas involution mammary matrix supported motility and invasion in highly metastatic MDA-MB-435 cells, but not in cells with low metastatic potential. Biochemical characterization of the matrices revealed intact fibronectin (FN) and low matrix metalloproteinase activity in nulliparous mammary matrix and fragmented FN and high matrix metalloproteinase activity in the matrix isolated from involuting glands. Purified intact FN was found to inhibit cell invasiveness, whereas FN fragments enhanced cell invasiveness in a matrix metalloproteinase-dependent manner. These data suggest that physiological changes that occur in the mammary extracellular matrix as a result of reproductive status alter the in vitro parameters of metastatic potential.

Mammary epithelial cell phagocytosis downstream of TGF-ß3 is characterized by adherens junction reorganization.

After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-ß (TGF-ß) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-ß enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-ß3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and ß-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or ß-catenin reduced the effect of TGF-ß3 on phagocytosis to near baseline levels. However, ß-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and ß-catenin signaling contribute to phagocytosis downstream of TGF-ß3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.

XactMice: humanizing mouse bone marrow enables microenvironment reconstitution in a patient-derived xenograft model of head and neck cancer.

The limitations of cancer cell lines have led to the development of direct patient-derived xenograft models. However, the interplay between the implanted human cancer cells and recruited mouse stromal and immune cells alters the tumor microenvironment and limits the value of these models. To overcome these constraints, we have developed a technique to expand human hematopoietic stem and progenitor cells (HSPCs) and use them to reconstitute the radiation-depleted bone marrow of a NOD/SCID/IL2rg(-/-) (NSG) mouse on which a patient's tumor is then transplanted (XactMice). The human HSPCs produce immune cells that home into the tumor and help replicate its natural microenvironment. Despite previous passage on nude mice, the expression of epithelial, stromal and immune genes in XactMice tumors aligns more closely to that of the patient tumor than to those grown in non-humanized mice-an effect partially facilitated by human cytokines expressed by both the HSPC progeny and the tumor cells. The human immune and stromal cells produced in the XactMice can help recapitulate the microenvironment of an implanted xenograft, reverse the initial genetic drift seen after passage on non-humanized mice and provide a more accurate tumor model to guide patient treatment.

Apoptosis in the genesis and prevention of cancer.

A strong rationale exists for developing cancer control strategies designed to suppress or reverse the development of precancerous lesions in order to reduce the occurrence and recurrence of this disease. Whereas numerous agents have been identified that inhibit tumor development, little is known about how they work. Recently the hypothesis has been raised that misregulation of apoptosis results in a failure of tissue size regulation that contributes to the development of cancer. If validated, this concept has important implications for the prevention of carcinogenesis and could lead to the development of new cancer control approaches that have as their basis the restoration of competence to regulate tissue size. Thus, it is essential to consider the role of cell loss in the tumorigenic process. In this regard investigation of the role of specific types of cell death in tumorigenesis, particularly the role of apoptotic cell death in maintenance of tissue homeostasis, has been neglected. The fact that apoptosis is a highly conserved, specific, and selective means of controlling tissue mass and shape also suggests that it can be exploited for the prevention or control of cancer.

Effect of methylated forms of selenium on cell viability and the induction of DNA strand breakage.

Selenobetaine (SB) and selenobetaine methyl ester (SBME) are methylated selenonium derivatives that undergo metabolism to release methyl selenide and dimethylselenide, respectively, as primary metabolites. Since methylation of selenium is considered to be detoxifying, the toxicologic activity of SB or SBME may differ from that of inorganic forms of selenium, such as selenite, that undergo reduction and can induce cell damage. In this study, the effects of SB, SBME and selenite on the viability and long-term growth potential of a mouse leukemia cell line (L1210) were compared. Treatment with 20 microM selenite reduced the rate of cell doubling and the long-term growth potential of cells as measured by colony-forming ability. These effects of selenite were accompanied by a reduction in DNA integrity, assessed by alkaline elution analysis for single-strand breaks. Exposure to 500 microM SB or SBME for 24 hr reduced the colony-forming ability of cells in the absence of any effect on dye exclusion or induction of single-strand breaks in DNA. Exposure of cells to 500 microM SB or SBME resulted in levels of intracellular selenium similar to those after exposure to 20 microM selenite. These observations indicate that it is possible to maintain high intracellular levels of selenium, by exposure to methylated selenocompounds, without affecting DNA integrity. These findings also suggest that DNA fragmentation resulting from exposure to selenite occurs during its reductive metabolism and not from the accumulation of a methylated metabolite of selenium. The fact that SB or SBME reduced the ability of L1210 cells to form colonies in agar in the absence of either DNA fragmentation or any effect on the ability of treated cells to exclude a vital dye suggests that both methylated compounds alter the long-term proliferative potential of cells via a mechanism(s) distinct from that associated with cell injury and death by necrosis. Efforts are underway to determine the origin of these effects.

Estrous cycle regulation of mammary epithelial cell proliferation, differentiation, and death in the Sprague-Dawley rat: a model for investigating the role of estrous cycling in mammary carcinogenesis.

The Sprague-Dawley rat is highly regarded for studies designed to investigate the effects of endocrine modulation on mammary carcinogenesis. In this study, we further evaluate the validity of the Sprague-Dawley rat model for the study of human breast cancer by evaluating the effects of normal 4-day estrous cycling on mammary epithelial cell proliferation, differentiation, and apoptotic death. Trends in mammary gland development with stage of 4-day estrous cycle were evident. Mammary glands isolated from follicular and early luteal stages had predominantly ductal histoarchitecture, whereas glands isolated from mid-late luteal were predominantly lobuloalveolar. Quantitation of BrdU incorporation revealed that epithelial cell proliferation was eight-fold higher in metestrus and diestrus-1 than in proestrus. Expression of beta-casein and whey acidic protein (WAP)4 mRNA was also highly dependent on stage of estrous, with detection restricted to midcycle. Apoptotic cell death of mammary epithelium was found to be suppressed during the peak in cell proliferation. TRPM-2/ clusterin mRNA was elevated when apoptosis was low and milk protein mRNA levels were high, consistent with putative roles for TRPM-2/clusterin in inhibiting cell death in regressing tissues and inducing mammary epithelial cell differentiation. Cell proliferation, differentiation, and death occurred only in a subset of epithelial cells per estrous cycle, and these cells appeared randomly distributed throughout multiple ductules and alveoli. These observations suggest that cellular response(s) to ovarian hormone-dependent signals is asynchronous. Cumulatively, these observations demonstrate that rat mammary epithelial cell proliferation, differentiation, and death are under the control of cycling ovarian hormones, similarly to the human mammary epithelium during the menstrual cycle.

Function of the her-1 gene is required for maintenance of male differentiation in adult tissues of C. elegans.

Function of the sex-determining gene her-1 is required in XO embryos of C. elegans to specify male development. Using a temperature-sensitive mutant of her-1, we show that when XO males reared at a permissive temperature are shifted as adults to a nonpermissive temperature, they initiate vitellogenin synthesis in the intestine and oocyte production in the germline. A similar shift has no effect on her-1(+) males. We conclude that sexual differentiation of the intestine and germline is plastic, requiring her-1 expression throughout adulthood for maintenance of the male state.

Autonomy and nonautonomy of sex determination in triploid intersex mosaics of C. elegans.

The primary sex-determining signal in Caenorhabditis elegans is the ratio of X chromosomes to sets of autosomes (X/A ratio), normally 1.0 in hermaphrodites (XX) and 0.5 in males (XO). XX triploids (X/A = 0.67) are males, but if these animals carry a partial duplication of the X chromosome such that X/A approximately equal to 0.7, they develop as intersexes that are sexually mosaic. We have analyzed these mosaics using Nomarski microscopy and in situ hybridization to obtain information on whether sex determination decisions can be made independently in different cells and tissues, and when these commitments are made. The observed patterns of male and female cells in individual animals indicate that sex determination decisions can be influenced by anterior-posterior position and that sex determination decisions can be made as late as the third larval stage of postembryonic development. Although these decisions clearly can be made independently in different lineages, they show substantial biases toward one sex or the other in individual animals. We interpret these results to suggest that sex determination in C. elegans is not entirely cell autonomous.

Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling.

Mammary gland form and function are regulated by interactions between epithelium and extracellular matrix. Major glycoprotein components of extracellular matrix have been identified that give survival, proliferation and differentiation signals to mammary epithelial cells. We provide evidence that proteolytic fragments of the extracellular matrix glycoprotein, fibronectin, suppress growth and can promote apoptosis of mouse mammary epithelial cells. During mammary gland involution, total fibronectin and fibronectin fragment levels are increased. The peak levels of fibronectin protein and fragments are observed 4-6 days post-weaning, coincident with the peak in epithelial cell death. Using a model for hormone withdrawal-induced death of mammary epithelium, elevated levels of fibronectin proteolytic fragments were associated with apoptosis in TM-6 cells, a tumorigenic mouse mammary epithelial cell line. Treatment of TM-6 cells with exogenous fibronectin fragments (FN120) reduced cell number, and induced apoptosis and matrix degrading protease activity. Inhibition of matrix protease activity rescued TM-6 cell viability, indicating that FN120-induced cell loss is mediated through matrix protease activity. In a three-dimensional model for mammary gland development, FN120 reduced alveolar-like and promoted ductal-like development by a matrix protease-dependent mechanism. These data suggest that during post-lactational involution, fibronectin fragments may contribute to epithelial cell loss and dissolution of mammary alveoli by inducing matrix degrading proteinases.

Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.

The effect of treatment with D-L-2-difluoromethylornithine (DFMO) plus retinyl acetate (RA) on the promotion stage of 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Combined treatment was more effective than single agent treatment in decreasing cancer incidence and multiplicity and in prolonging cancer latency. Increased efficacy was associated with reduced morphological complexity of the gland and increased mammary extracellular matrix. Using ovarian hormones to model mitogen stimulation in the mammary gland, DFMO plus RA treatment reduced mammary gland complexity in the absence of an effect on bromodeoxyuridine (BrDU) labeling index measured immunohistochemically. Morphological and biochemical evaluation of these glands revealed increased levels of extracellular matrix in rats treated with chemopreventive agents. Tenascin expression and fibronectin levels were elevated and laminin levels were decreased. The fact that matrix degrading proteinase activity was also increased indicated that tissue remodeling was modulated by these chemopreventive agents. These data provide evidence of alterations in epithelial cell-extracellular matrix interactions that could account in part for the chemopreventive effects of DFMO plus RA.