Pepper Schedin

Mammary gland ECM remodeling, stiffness, and mechanosignaling in normal development and tumor progression.

Cells of the mammary gland are in intimate contact with other cells and with the extracellular matrix (ECM), both of which provide not only a biochemical context, but a mechanical context as well. Cell-mediated contraction allows cells to sense the stiffness of their microenvironment, and respond with appropriate mechanosignaling events that regulate gene expression and differentiation. ECM composition and organization are tightly regulated throughout development of the mammary gland, resulting in corresponding regulation of the mechanical environment and proper tissue architecture. Mechanical regulation is also at play during breast carcinoma progression, as changes in ECM deposition, composition, and organization accompany breast carcinoma. These changes result in stiffer matrices that activate mechanosignaling pathways and thereby induce cell proliferation, facilitate local tumor cell invasion, and promote progression. Thus, understanding the role of forces in the mammary gland is crucial to understanding both normal developmental and pathological processes.

Pregnancy-associated breast cancer and metastasis

Pregnancy-associated breast cancer, which has a poor prognosis, is often overlooked by clinicians and researchers alike. With the trend towards delayed child-bearing, an increase in the occurrence of breast cancer complicated by pregnancy is anticipated. The mechanisms that have been proposed to account for this poor prognosis, including increased hormone exposure, might not contribute significantly to the observed increase in metastasis seen in these patients. Instead, the mammary microenvironment might become tumour-promoting after pregnancy because of the remodelling of the mammary gland to its pre-pregnant state. This remodelling, which is associated with pro-inflammatory and wound-healing mechanisms, is proposed to support tumour-cell dissemination. This hypothesis will be discussed.

Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2

The prognosis of breast cancer in young women is influenced by reproductive history. Women diagnosed within 5 years postpartum have worse prognosis than nulliparous women or women diagnosed during pregnancy. Here we describe a mouse model of postpartum breast cancer that identifies mammary gland involution as a driving force of tumor progression. In this model, human breast cancer cells exposed to the involuting mammary microenvironment form large tumors that are characterized by abundant fibrillar collagen, high cyclooxygenase-2 (COX-2) expression and an invasive phenotype. In culture, tumor cells are invasive in a fibrillar collagen and COX-2–dependent manner. In the involuting mammary gland, inhibition of COX-2 reduces the collagen fibrillogenesis associated with involution, as well as tumor growth and tumor cell infiltration to the lung. These data support further research to determine whether women at high risk for postpartum breast cancer would benefit from treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) during postpartum involution.

Alternatively Activated Macrophages and Collagen Remodeling Characterize the Postpartum Involuting Mammary Gland across Species

Recent pregnancy correlates with decreased survival for breast cancer patients compared with non-pregnancy-associated breast cancer. We hypothesize that postpartum mammary involution induces metastasis through wound-healing programs known to promote cancer. It is unknown whether alternatively activated M2 macrophages, immune cells important in wound-healing and experimental tumorigenesis that also predict poor prognosis for breast cancer patients, are recruited to the normal involuting gland. Macrophage markers CD68, CSF-1R, and F4/80 were examined across the pregnancy and involution cycle in rodent and human mammary tissues. Quantitative immunohistochemistry revealed up to an eightfold increase in macrophage number during involution, which returned to nulliparous levels with full regression. The involution macrophages exhibit an M2 phenotype as determined by high arginase-1 and low inducible nitric oxide synthase staining in rodent tissue, and by mannose receptor expression in human breast tissue. M2 cytokines IL-4 and IL-13 also peaked during involution. Extracellular matrix (ECM) isolated from involuting rat mammary glands was chemotactic for macrophages compared with nulliparous mammary ECM. Fibrillar collagen levels and proteolysis increased dramatically during involution, and denatured collagen I acted as a strong chemoattractant for macrophages in cell culture, suggesting proteolyzed fibrillar collagen as a candidate ECM mediator of macrophage recruitment. M2 macrophages, IL-4, IL-13, fibrillar collagen accumulation, and proteolysis of collagen are all components of tumor promotional microenvironments, and thus may mediate promotion of breast cancers arising in the postpartum setting.

Pregnancy and Breast Cancer: when They Collide

Women of childbearing age experience an increased breast cancer risk associated with a completed pregnancy. For younger women, this increase in breast cancer risk is transient and within a decade after parturition a cross over effect results in an ultimate protective benefit. The post-partum peak of increased risk is greater in women with advanced maternal age. Further, their lifetime risk for developing breast cancer remains elevated for many years, with the cross over to protection occurring decades later or not at all. Breast cancers diagnosed during pregnancy and within a number of years post-partum are termed pregnancy-associated or PABC. Contrary to popular belief, PABC is not a rare disease and could affect up to 40,000 women in 2009. The collision between pregnancy and breast cancer puts women in a fear-invoking paradox of their own health, their pregnancy, and the outcomes for both. We propose two distinct subtypes of PABC: breast cancer diagnosed during pregnancy and breast cancer diagnosed post-partum. This distinction is important because emerging epidemiologic data highlights worsened outcomes specific to post-partum cases. We reported that post-partum breast involution may be responsible for the increased metastatic potential of post-partum PABC. Increased awareness and detection, rationally aggressive treatment, and enhanced understanding of the mechanisms are imperative steps toward improving the prognosis for PABC. If we determine the mechanisms by which involution promotes metastasis of PABC, the post-partum period can be a window of opportunity for intervention strategies.

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 β-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.

Mammary ECM composition and function are altered by reproductive state.

To address whether reproductive state alters mammary gland extracellular matrix (ECM) composition and function, ECM was isolated from nulliparous, pregnant, lactating, involuting, and regressed rat mammary glands. The ECM composition of fibronectin, tenascin, laminin, clusterin, and MMPs was found to vary dramatically with reproductive state. In 3‐dimensional (3‐D) culture, we identified novel effects of these endogenous mammary matrices on mammary epithelial cells. Specifically we found that (1) matrix isolated from nulliparous animals promoted the formation of epithelial ducts with bifurcation, (2) matrix isolated from mid‐involuting mammary glands induced cell death, (3) matrix isolated from late‐stage involuting glands restored glandular development, while (4) matrix isolated from parous animals restricted glandular morphogenesis. Our data were consistent with mammary gland ECM facilitating epithelial cell proliferation, differentiation, death, and glandular reorganization that occur during the pregnancy and involution cycle. Further, we show that the parous gland has persistent changes in ECM function. Cumulatively, our data demonstrated that the microenvironment of the normal adult mammary gland is highly plastic, which has important implications for mammary tumor cell progression and dormancy. These data also raised the possibility of targeting mammary matrix production with preventive or therapeutic interventions.

Microenvironment of the Involuting Mammary Gland Mediates Mammary Cancer Progression

Breast cancer diagnosed after a completed pregnancy has higher metastatic potential and therefore a much poorer prognosis. We hypothesize that following pregnancy the process of mammary gland involution, which returns the gland to its pre-pregnant state, co-opts some of the programs of wound healing. The pro-inflammatory milieu that results, while physiologically normal, promotes tumor progression. In this review, the similarities between mammary gland involution after cessation of milk-production and pathological tissue remodeling are discussed in light of emerging data demonstrating a role for pathological tissue remodeling in cancer.

Multistep tumorigenesis and the microenvironment

Early-stage cancers have long been considered to be less aggressive than late-stage cancers because it is assumed that they have accumulated fewer of the mutations that are required for full metastatic potential. For breast cancer, recent gene expression profiling data have challenged this paradigm by identifying early-stage cancers with similar gene expression profiles to fully metastatic cancers. In this review, multistep carcinogenesis is reconsidered in light of these new data. The concept that the tumor stroma plays a key role in determining whether a metastatic tumor cell will remain dormant or become invasive is discussed. Recent studies demonstrating the feasibility of targeting tumor stroma for cancer prevention and treatment are presented.

Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution

Mammary gland development is dependent on macrophages, as demonstrated by their requirement during the expansion phases of puberty and pregnancy. Equally dramatic tissue restructuring occurs following lactation, when the gland regresses to a state that histologically resembles pre-pregnancy through massive programmed epithelial cell death and stromal repopulation. Postpartum involution is characterized by wound healing-like events, including an influx of macrophages with M2 characteristics. Macrophage levels peak after the initial wave of epithelial cell death, suggesting that initiation and execution of cell death are macrophage independent. To address the role of macrophages during weaning-induced mammary gland involution, conditional systemic deletion of macrophages expressing colony stimulating factor 1 receptor (CSF1R) was initiated just prior to weaning in the Mafia mouse model. Depletion of CSF1R+ macrophages resulted in delayed mammary involution as evidenced by loss of lysosomal-mediated and apoptotic epithelial cell death, lack of alveolar regression and absence of adipocyte repopulation 7 days post-weaning. Failure to execute involution occurred in the presence of milk stasis and STAT3 activation, indicating that neither is sufficient to initiate involution in the absence of CSF1R+ macrophages. Injection of wild-type bone marrow-derived macrophages (BMDMs) or M2-differentiated macrophages into macrophage-depleted mammary glands was sufficient to rescue involution, including apoptosis, alveolar regression and adipocyte repopulation. BMDMs exposed to the postpartum mammary involution environment upregulated the M2 markers arginase 1 and mannose receptor. These data demonstrate the necessity of macrophages, and implicate M2-polarized macrophages, for epithelial cell death during normal postpartum mammary gland involution.