Chitin-mediated blockade of chitinase-like proteins reduces tumor immunosuppression, inhibits lymphatic metastasis and enhances anti-PD-1 efficacy in complementary TNBC models.

BACKGROUND: Chitinase-like proteins (CLPs) play a key role in immunosuppression under inflammatory conditions such as cancer. CLPs are enzymatically inactive and become neutralized upon binding of their natural ligand chitin, potentially reducing CLP-driven immunosuppression. We investigated the efficacy of chitin treatment in the context of triple-negative breast cancer (TNBC) using complementary mouse models. We also evaluated the immunomodulatory influence of chitin on immune checkpoint blockade (ICB) and compared its efficacy as general CLP blocker with blockade of a single CLP, i.e. chitinase 3-like 1 (CHI3L1). METHODS: Female BALB/c mice were intraductally injected with luciferase-expressing 4T1 or 66cl4 cells and systemically treated with chitin in combination with or without anti-programmed death (PD)-1 ICB. For single CLP blockade, tumor-bearing mice were treated with anti-CHI3L1 antibodies. Metastatic progression was monitored through bioluminescence imaging. Immune cell changes in primary tumors and lymphoid organs (i.e. axillary lymph nodes and spleen) were investigated through flow cytometry, immunohistochemistry, cytokine profiling and RNA-sequencing. CHI3L1-stimulated RAW264.7 macrophages were subjected to 2D lymphatic endothelial cell adhesion and 3D lymphatic integration in vitro assays for studying macrophage-mediated lymphatic remodeling. RESULTS: Chitin significantly reduced primary tumor progression in the 4T1-based model by decreasing the high production of CLPs that originate from tumor-associated neutrophils (TANs) and Stat3 signaling, prominently affecting the CHI3L1 and CHI3L3 primary tumor levels. It reduced immunosuppressive cell types and increased anti-tumorigenic T-cells in primary tumors as well as axillary lymph nodes. Chitin also significantly reduced CHI3L3 primary tumor levels and immunosuppression in the 66cl4-based model. Compared to anti-CHI3L1, chitin enhanced primary tumor growth reduction and anti-tumorigenicity. Both treatments equally inhibited lymphatic adhesion and integration of macrophages, thereby hampering lymphatic tumor cell spreading. Upon ICB combination therapy, chitin alleviated anti-PD-1 resistance in both TNBC models, providing a significant add-on reduction in primary tumor and lung metastatic growth compared to chitin monotherapy. These add-on effects occurred through additional increase in CD8α+ T-cell infiltration and activation in primary tumor and lymphoid organs. CONCLUSIONS: Chitin, as a general CLP blocker, reduces CLP production, enhances anti-tumor immunity as well as ICB responses, supporting its potential clinical relevance in immunosuppressed TNBC patients.

Toward Characterizing Lymphatic Vasculature in the Mammary Gland During Normal Development and Tumor-Associated Remodeling.

Lymphatic vasculature has been shown to promote metastatic spread of breast cancer. Lymphatic vasculature, which is made up of larger collecting vessels and smaller capillaries, has specialized cell junctions that facilitate cell intravasation. Normally, these junctions are designed to collect immune cells and other cellular components for immune surveillance by lymph nodes, but they are also utilized by cancer cells to facilitate metastasis. Although lymphatic development overall in the body has been well-characterized, there has been little focus on how the lymphatic network changes in the mammary gland during stages of remodeling such as pregnancy, lactation, and postpartum involution. In this review, we aim to define the currently known lymphangiogenic factors and lymphatic remodeling events during mammary gland morphogenesis. Furthermore, we juxtapose mammary gland pubertal development and postpartum involution to show similarities of pro-lymphangiogenic signaling as well as other molecular signals for epithelial cell survival that are critical in these morphogenic stages. The similar mechanisms include involvement of M2-polarized macrophages that contribute to matrix remodeling and vasculogenesis; signal transducer and activator of transcription (STAT) survival and proliferation signaling; and cyclooxygenase 2 (COX2)/Prostaglandin E2 (PGE2) signaling to promote ductal and lymphatic expansion. Investigation and characterization of lymphangiogenesis in the normal mammary gland can provide insight to targetable mechanisms for lymphangiogenesis and lymphatic spread of tumor cells in breast cancer.

Breast cancers co-opt normal mechanisms of tolerance to promote immune evasion and metastasis.

Normal developmental processes, such as those seen during embryonic development and postpartum mammary gland involution, can be reactivated by cancer cells to promote immune suppression, tumor growth, and metastatic spread. In mammalian embryos, paternal-derived antigens are at risk of being recognized as foreign by the maternal immune system. Suppression of the maternal immune response toward the fetus, which is mediated in part by the trophoblast, is critical to ensure embryonic survival and development. The postpartum mammary microenvironment also exhibits immunosuppressive mechanisms accompanying the massive cell death and tissue remodeling that occurs during mammary gland involution. These normal immunosuppressive mechanisms are paralleled during malignant transformation, where tumors can develop neoantigens that may be recognized as foreign by the immune system. To circumvent this, tumors can dedifferentiate and co-opt immune-suppressive mechanisms normally utilized during fetal tolerance and postpartum mammary involution. In this review, we discuss those similarities and how they can inform our understanding of cancer progression and metastasis.

GPR182 limits antitumor immunity via chemokine scavenging in mouse melanoma models.

For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.

Triple Targeting of Breast Tumors Driven by Hormonal Receptors and HER2.

Breast cancers that express hormonal receptors (HR) and HER2 display resistance to targeted therapy. Tumor-promotional signaling from the HER2 and estrogen receptor (ER) pathways converges at the cyclin D1 and cyclin-dependent kinases (CDK) 4 and 6 complex, which drives cell-cycle progression and development of therapeutic resistance. Therefore, we hypothesized that co-targeting of ER, HER2, and CDK4/6 may result in improved tumoricidal activity and suppress drug-resistant subclones that arise on therapy. We tested the activity of the triple targeted combination therapy with tucatinib (HER2 small-molecule inhibitor), palbociclib (CKD4/6 inhibitor), and fulvestrant (selective ER degrader) in HR+/HER2+ human breast tumor cell lines and xenograft models. In addition, we evaluated whether triple targeted combination prevents growth of tucatinib or palbociclib-resistant subclones in vitro and in vivo Triple targeted combination significantly reduced HR+/HER2+ tumor cell viability, clonogenic survival, and in vivo growth. Moreover, survival of HR+/HER2+ cells that were resistant to the third drug in the regimen was reduced by the other two drugs in combination. We propose that a targeted triple combination approach will be clinically effective in the treatment of otherwise drug-resistant tumors, inducing robust responses in patients.

Anoikis resistance in mammary epithelial cells is mediated by semaphorin 7a.

Semaphorin-7a (SEMA7A), best known as a neuroimmune molecule, plays a diverse role in many cellular processes and pathologies. Here, we show that SEMA7A promotes anoikis resistance in cultured mammary epithelial cells through integrins and activation of pro-survival kinase AKT, which led us to investigate a role for SEMA7A during postpartum mammary gland involution-a normal developmental process where cells die by anoikis. Our results reveal that SEMA7A is expressed on live mammary epithelial cells during involution, that SEMA7A expression is primarily observed in α6-integrin expressing cells, and that luminal progenitor cells, specifically, are decreased in mammary glands of SEMA7A-/- mice during involution. We further identify a SEMA7A-α6/ß1-integrin dependent mechanism of mammosphere formation and chemoresistance in mammary epithelial cells and suggest that this mechanism is relevant for recurrence in breast cancer patients.

Rab40-Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration.

Rab40b is a SOCS box-containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b-Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b-Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b-Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

Hormonal Regulation of Semaphorin 7a in ER+ Breast Cancer Drives Therapeutic Resistance.

Approximately 70% of all breast cancers are estrogen receptor-positive (ER+ breast cancer), and endocrine therapy has improved survival for patients with ER+ breast cancer. However, up to half of these tumors recur within 20 years. Recurrent ER+ breast cancers develop resistance to endocrine therapy; thus, novel targets are needed to treat recurrent ER+ breast cancer. Here we report that semaphorin 7A (SEMA7A) confers significantly decreased patient survival rates in ER+ breast cancer. SEMA7A was hormonally regulated in ER+ breast cancer, but its expression did not uniformly decrease with antiestrogen treatments. Additionally, overexpression of SEMA7A in ER+ cell lines drove increased in vitro growth in the presence of estrogen deprivation, tamoxifen, and fulvestrant. In vivo, SEMA7A conferred primary tumor resistance to fulvestrant and induced lung metastases. Prosurvival signaling was identified as a therapeutic vulnerability of ER+SEMA7A+ tumors. We therefore propose that targeting this pathway with inhibitors of survival signaling such as venetoclax may prove efficacious for treating SEMA7A+ tumors. SIGNIFICANCE: SEMA7A predicts for and likely contributes to poor response to standard-of-care therapies, suggesting that patients with SEMA7A+ER+ tumors may benefit from alternative therapeutic strategies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/1/187/F1.large.jpg.

Extracellular vesicles from young women's breast cancer patients drive increased invasion of non-malignant cells via the Focal Adhesion Kinase pathway: a proteomic approach.

BACKGROUND: Extracellular vesicles (EVs) are small membrane particles that contribute to cancer progression and metastases by transporting biologically significant proteins and nucleic acids. They may also serve as biomarkers of various disease states or important therapeutic targets. Breast cancer EVs have the potential to change the behavior of other cells in their microenvironment. However, the proteomic content of EVs isolated from young women's breast cancer patients and the mechanisms underlying the influence of EVs on tumor cell behavior have not yet been reported. METHODS: In our current translational studies, we compared the proteomic content of EVs isolated from invasive breast cancer cell lines and plasma samples from young women's breast cancer (YWBC) patients and age-matched healthy donors using mass spectrometry. We analyzed the functionality of EVs in two dimensional tumor cell invasion assays and the gene expression changes in tumor cells after incubation with EVs. RESULTS: We found that treatment with EVs from both invasive breast cancer cell lines and plasma of YWBC patients altered the invasive properties of non-invasive breast cancer cells. Proteomics identified differences between EVs from YWBC patients and healthy donors that correlated with their altered function. Further, we identified gene expression changes in non-invasive breast cancer cells after treatment with EVs that implicate the Focal Adhesion Kinase (FAK) signaling pathway as a potential targetable pathway affected by breast cancer-derived EVs. CONCLUSIONS: Our results suggest that the proteome of EVs from breast cancer patients reflects their functionality in tumor motility assays and may help elucidate the role of EVs in breast cancer progression.

Semaphorin 7a is a biomarker for recurrence in postpartum breast cancer.

Breast cancer is a global health threat and cases diagnosed in women during the years after childbirth, or postpartum breast cancers (PPBCs), have high risk for metastasis. In preclinical murine models, semaphorin 7a (SEMA7A) drives the metastatic potential of postpartum mammary tumors. Thus, we hypothesize that SEMA7A may drive metastasis of PPBC in women. We report that SEMA7A protein expression is increased in PPBCs compared to their nulliparous counterparts in our University of Colorado cohort. Additionally, tumors from PPBC patients with involved lymph nodes and lymphovascular invasion were higher on average suggesting a potential role for SEMA7A as a prognostic biomarker. Consistent with this hypothesis we identify a level of SEMA7A expression in tumors that can predict for recurrence. We propose SEMA7A as a potential biomarker and therapeutic target for PPBC patients, who currently lack strong predictors of outcome and unique targeted therapy options.

Cancer Cell CD44 Mediates Macrophage/Monocyte-Driven Regulation of Head and Neck Cancer Stem Cells.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

Macphatics and PoEMs in Postpartum Mammary Development and Tumor Progression.

Postpartum mammary gland involution is a mammalian tissue remodeling event that occurs after pregnancy and lactation to return the gland to the pre-pregnant state. This event is characterized by apoptosis and lysosomal-mediated cell death of the majority of the lactational mammary epithelium, followed by remodeling of the extracellular matrix, influx of immune cell populations (in particular, T helper cells, monocytes, and macrophages), and neo-lymphangiogenesis. This postpartum environment has been shown to be promotional for tumor growth and metastases and may partially account for why women diagnosed with breast cancer during the postpartum period or within 5 years of last childbirth have an increased risk of developing metastases when compared to their nulliparous counterparts. The lymphatics and macrophages present during mammary gland involution have been implicated in promoting the observed growth and metastasis. Of importance are the macrophages, which are of the "M2" phenotype and are known to create a pro-tumor microenvironment. In this report, we describe a subset of postpartum macrophages that express lymphatic proteins (PoEMs) and directly interact with lymphatic vessels to form chimeric vessels or "macphatics". Additionally, these PoEMs are very similar to tumor-associated macrophages that also express lymphatic proteins and are present at the sites of lymphatic vessels where tumors escape the tissue and enter the lymphatic vasculature. Further characterizing these PoEMs may offer insight in preventing lymphatic metastasis of breast cancer, as well as provide information for how developmental programming of lymphatic endothelial cells and macrophages can contribute to different disease progression.

Postpartum Involution and Cancer: An Opportunity for Targeted Breast Cancer Prevention and Treatments?

Childbirth at any age confers a transient increased risk for breast cancer in the first decade postpartum and this window of adverse effect extends over two decades in women with late-age first childbirth (>35 years of age). Crossover to the protective effect of pregnancy is dependent on age at first pregnancy, with young mothers receiving the most benefit. Furthermore, breast cancer diagnosis during the 5- to 10-year postpartum window associates with high risk for subsequent metastatic disease. Notably, lactation has been shown to be protective against breast cancer incidence overall, with varying degrees of protection by race, multiparity, and lifetime duration of lactation. An effect for lactation on breast cancer outcome after diagnosis has not been described. We discuss the most recent data and mechanistic insights underlying these epidemiologic findings. Postpartum involution of the breast has been identified as a key mediator of the increased risk for metastasis in women diagnosed within 5-10 years of a completed pregnancy. During breast involution, immune avoidance, increased lymphatic network, extracellular matrix remodeling, and increased seeding to the liver and lymph node work as interconnected pathways, leading to the adverse effect of a postpartum diagnosis. We al discuss a novel mechanism underlying the protective effect of breastfeeding. Collectively, these mechanistic insights offer potential therapeutic avenues for the prevention and/or improved treatment of postpartum breast cancer.

Postpartum breast cancer progression is driven by semaphorin 7a-mediated invasion and survival.

Young women diagnosed with breast cancer (BC) have poor prognosis due to increased rates of metastasis. In addition, women diagnosed within 10 years of most recent childbirth are approximately three times more likely to develop metastasis than age- and stage-matched nulliparous women. We define these cases as postpartum BC (PPBC) and propose that the unique biology of the postpartum mammary gland drives tumor progression. Our published results revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor-associated lymphangiogenesis, all of which are also increased in preclinical models of PPBC. However, whether SEMA7A drives progression in PPBC remains largely unexplored. Our results presented herein show that silencing of SEMA7A decreases tumor growth in a model of PPBC, while overexpression is sufficient to increase growth in nulliparous hosts. Further, we show that SEMA7A promotes multiple known drivers of PPBC progression including tumor-associated COX-2 expression and fibroblast-mediated collagen deposition in the tumor microenvironment. In addition, we show for the first time that SEMA7A-expressing cells deposit fibronectin to promote tumor cell survival. Finally, we show that co-expression of SEMA7A/COX-2/FN predicts for poor prognosis in breast cancer patient cohorts. These studies suggest SEMA7A as a key mediator of BC progression, and that targeting SEMA7A may open avenues for novel therapeutic strategies.

Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer.

BACKGROUND: Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2. METHODS: For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis. RESULTS: Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in re-expression of SIM2. CONCLUSION: Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.

PD-1 Blockade During Post-partum Involution Reactivates the Anti-tumor Response and Reduces Lymphatic Vessel Density.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ~3-5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNγ and TNFα. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

Studies of postpartum mammary gland involution reveal novel pro-metastatic mechanisms.

Postpartum involution is the process by which the lactating mammary gland returns to the pre-pregnant state after weaning. Expression of tumor-promotional collagen, upregulation of matrix metalloproteinases, infiltration of M2 macrophages, and remodeling of blood and lymphatic vasculature are all characteristics shared by the involuting mammary gland and breast tumor microenvironment. The tumor promotional nature of the involuting mammary gland is perhaps best evidenced by cases of postpartum breast cancer (PPBC), or those cases diagnosed within 10 years of most recent childbirth. Women with PPBC experience more aggressive disease and higher risk of metastasis than nulliparous patients and those diagnosed outside the postpartum window. Semaphorin 7a (SEMA7A), cyclooxygenase-2 (COX-2), and collagen are all expressed in the involuting mammary gland and, together, predict for decreased metastasis free survival in breast cancer. Studies investigating the role of these proteins in involution have been important for understanding their contributions to PPBC. Postpartum involution thus represents a valuable model for the identification of novel molecular drivers of PPBC and classical cancer hallmarks. In this review, we will highlight the similarities between involution and cancer in the mammary gland, and further define the contribution of SEMA7A/COX-2/collagen interplay to postpartum involution and breast tumor progression and metastasis.

Association Between Postpartum Breast Cancer Diagnosis and Metastasis and the Clinical Features Underlying Risk.

Importance: In women 45 years or younger, breast cancer diagnosis after childbirth increases the risk for metastasis and death, yet limited data exist to define this window of risk and associated prognostic factors. Objective: To assess the window of elevated risk for metastasis following a postpartum breast cancer (PPBC) diagnosis and whether clinical prognostic factors are associated with the increased risk. Design, Setting, and Participants: This multicenter cohort study conducted using cases from the Colorado Young Women's Breast Cancer Cohort diagnosed between January 1, 1981, and December 31, 2014, included 701 women 45 years or younger with stage I to III invasive breast cancer for whom parity data, including time of last childbirth, were available. Data analysis was conducted from July 1 to September 30, 2017. This study involved a tertiary care academic hospital-based breast center and its regional affiliates with cases from the greater Rocky Mountain region. Exposures: Primary exposures were prior childbirth or no childbirth, time between most recent childbirth and breast cancer diagnosis, and time between breast cancer diagnosis and metastasis. Main Outcomes and Measures: The primary outcome was distant metastasis-free survival. Results: A total of 701 women 45 years or younger from the greater Rocky Mountain states region were included in the analysis; mean (SD) age at diagnosis was 37.9 (5.1) years. Breast cancer diagnosis within 10 years after parturition was associated with elevated risk for metastasis, particularly in women with stage I or II disease. In addition, women with PPBC diagnosed within 10 years of a completed pregnancy that was estrogen receptor-positive showed distant metastasis-free survival similar to that of nulliparous patients with estrogen receptor-negative cancer, and women with estrogen receptor-negative PPBC had further reduced metastasis-free survival. Moreover, women with PPBC had increased lymphovascular invasion and lymph node involvement. In addition, tumor-associated Ki67 positivity identified 129 patients with luminal B cancer in the cohort that, independent of parity status, had poorer prognosis compared with patients with luminal A cancer, although it did not reach statistical significance. Conclusions and Relevance: Diagnosis of PPBC within 10 years post partum appears to be associated with an increased risk for metastasis. This increased risk was highest in stages I and II cancer at diagnosis and present in both patients with estrogen receptor-positive and estrogen receptor-negative cancer, persisting in estrogen receptor-positive cases for up to 15 years after diagnosis. Postpartum breast cancer diagnoses were not associated with increased Ki67 index but were associated with increased lymphovascular invasion and lymph node involvement compared with breast cancer in nulliparous patients.

Semaphorin 7A Promotes Macrophage-Mediated Lymphatic Remodeling during Postpartum Mammary Gland Involution and in Breast Cancer.

Postpartum mammary gland involution is a tissue remodeling event that occurs in all mammals in the absence of nursing or after weaning to return the gland to the pre-pregnant state. The tissue microenvironment created by involution has proven to be tumor promotional. Here we report that the GPI-linked protein semaphorin 7A (SEMA7A) is expressed on mammary epithelial cells during involution and use preclinical models to demonstrate that tumors induced during involution express high levels of SEMA7A. Overexpression of SEMA7A promoted the presence of myeloid-derived podoplanin (PDPN)-expressing cells in the tumor microenvironment and during involution. SEMA7A drove the expression of PDPN in macrophages, which led to integrin- and PDPN-dependent motility and adherence to lymphatic endothelial cells to promote lymphangiogenesis. In support of this mechanism, mammary tissue from SEMA7A-knockout mice exhibited decreased myeloid-derived PDPN-expressing cells, PDPN-expressing endothelial cells, and lymphatic vessel density. Furthermore, coexpression of SEMA7A, PDPN, and macrophage marker CD68 predicted for decreased distant metastasis-free survival in a cohort of over 600 cases of breast cancer as well as in ovarian, lung, and gastric cancers. Together, our results indicate that SEMA7A may orchestrate macrophage-mediated lymphatic vessel remodeling, which in turn drives metastasis in breast cancer.Signficance: SEMA7A, which is expressed on mammary cells during glandular involution, alters macrophage biology and lymphangiogenesis to drive breast cancer metastasis. Cancer Res; 78(22); 6473-85. ©2018 AACR.