Traci R. Lyons

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.

Poliovirus 5′-Terminal Cloverleaf RNA Is Required in cis for VPg Uridylylation and the Initiation of Negative-Strand RNA Synthesis

ABSTRACT Chimeric poliovirus RNAs, possessing the 5′ nontranslated region (NTR) of hepatitis C virus in place of the 5′ NTR of poliovirus, were used to examine the role of the poliovirus 5′ NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3′ NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5′-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5′-terminal cloverleaf and 3′ NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA.

Collagen architecture in pregnancy-induced protection from breast cancer

Summary The reduction in breast cancer risk attributed to early-age pregnancy is mediated in part by changes in the mammary epithelium. Here, we address the role of the mammary stroma in this protection. Utilizing tumor cells capable of transitioning from indolent to proliferative or invasive states, we demonstrate that mammary extracellular matrix (ECM) from parous rats (parous matrix) decreases tumor growth and impedes cellular phenotypes associated with tumor cell invasion compared with that observed using nulliparous matrix. Proteomic analysis identifies an increased abundance of collagen I in parous matrix, an observation extended to breast tissue of parous women. Given the pro-tumorigenic attributes of fibrillar collagen, these results were unexpected. Second-harmonic generation imaging and atomic force microscopy revealed that the abundant collagen observed in the mammary glands of parous rats is less linearized and associated with a decrease in stromal stiffness, implicating collagen organization and stiffness in parity-induced protection. Using 3D cell culture models, we demonstrate that linearized (fibrillar) collagen I induces cellular phenotypes consistent with an invasive behavior in mammary tumor cells and alters the subcellular distribution of &bgr;1 integrin. Conversely, high-density non-fibrillar collagen I induces tumor-suppressive attributes, including increases in junctional E-cadherin in tumor cells, upregulation of genes encoding components of cell–cell junctions, and downregulation of mesenchymal-specific and metalloproteinase-encoding genes. These data show that collagen organization, rather than density alone, is a key contributor to the invasive phenotype. Furthermore, our data show that parity alters the composition and organization of mammary ECM, particularly fibrillar collagen, in a manner consistent with tumor suppression.

Tamoxifen induces pleiotrophic changes in mammary stroma resulting in extracellular matrix that suppresses transformed phenotypes

IntroductionThe functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma. To test this hypothesis, the question was addressed of whether mammary stroma is functionally altered by the anti-oestrogen drug tamoxifen.MethodsForty female rats at 70 days of age were randomised to two groups of 20 and treated with 1.0 mg/kg tamoxifen or vehicle subcutaneously daily for 30 days, followed by a three-day wash out period. Mammary tissue was harvested and effects of tamoxifen on mammary epithelium and stroma determined.ResultsAs expected, tamoxifen suppressed mammary alveolar development and mammary epithelial cell proliferation. Primary mammary fibroblasts isolated from tamoxifen-treated rats displayed a three-fold decrease in motility and incorporated less fibronectin in their substratum in comparison to control fibroblasts; attributes indicative of fibroblast quiescence. Immunohistochemistry analysis of CD68, a macrophage lysosomal marker, demonstrated a reduction in macrophage infiltration in mammary glands of tamoxifen-treated rats. Proteomic analyses by mass spectrometry identified several extracellular matrix (ECM) proteins with expression levels with tamoxifen treatment that were validated by Western blot. Mammary tissue from tamoxifen-treated rats had decreased fibronectin and increased collagen 1 levels. Further, ECM proteolysis was reduced in tamoxifen-treated rats as detected by reductions in fibronectin, laminin 1, laminin 5 and collagen 1 cleavage fragments. Consistent with suppression in ECM proteolysis with tamoxifen treatment, matrix metalloproteinase-2 levels and activity were decreased. Biochemically extracted mammary ECM from tamoxifen-treated rats suppressed in vitro macrophage motility, which was rescued by the addition of proteolysed collagen or fibronectin. Mammary ECM from tamoxifen-treated rats also suppressed breast tumour cell motility, invasion and haptotaxis, reduced organoid size in 3-dimensional culture and blocked tumour promotion in an orthotopic xenograft model; effects which could be partially reversed by the addition of exogenous fibronectin.ConclusionsThese data support the hypothesis that mammary stroma responds to tamoxifen treatment in concert with the epithelium and remodels to a microenvironment inhibitory to tumour cell progression. Reduced fibronectin levels and reduced ECM turnover appear to be hallmarks of the quiescent mammary microenvironment. These data may provide insight into attributes of a mammary microenvironment that facilitate tumour dormancy.

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.

Cyclooxygenase-2-dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer.

Breast involution following pregnancy has been implicated in the high rates of metastasis observed in postpartum breast cancers; however, it is not clear how this remodeling process promotes metastasis. Here, we demonstrate that human postpartum breast cancers have increased peritumor lymphatic vessel density that correlates with increased frequency of lymph node metastases. Moreover, lymphatic vessel density was increased in normal postpartum breast tissue compared with tissue from nulliparous women. In rodents, mammary lymphangiogenesis was upregulated during weaning-induced mammary gland involution. Furthermore, breast cancer cells exposed to the involuting mammary microenvironment acquired prolymphangiogenic properties that contributed to peritumor lymphatic expansion, tumor size, invasion, and distant metastases. Finally, in rodent models of postpartum breast cancer, cyclooxygenase-2 (COX-2) inhibition during the involution window decreased normal mammary gland lymphangiogenesis, mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatics, and metastasis. Our data indicate that physiologic COX-2-dependent lymphangiogenesis occurs in the postpartum mammary gland and suggest that tumors within this mammary microenvironment acquire enhanced prolymphangiogenic activity. Further, our results suggest that the prolymphangiogenic microenvironment of the postpartum mammary gland has potential as a target to inhibit metastasis and suggest that further study of the therapeutic efficacy of COX-2 inhibitors in postpartum breast cancer is warranted.

Regulation of the Pro-apoptotic scaffolding protein POSH by Akt.

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival.

AKT regulates BRCA1 stability in response to hormone signaling.

The observation that inherited mutations within BRCA1 result in breast and ovarian cancers suggests a functional relationship may exist between hormone signaling and BRCA1 function. We demonstrate that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling, and the rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis. Further, we identify a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. These data suggest AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1s role in DNA repair. Targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for breast and ovarian cancer treatment.