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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.

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.

Supplemental methods, figures, and table.

Macrophage gating strategy and chemoattractant expression levels

Oncomine analysis of SEMA7A expression with ER/PR biomarker status, grade, and stage

Generation of fulvestrant resistant cells and gating strategy for flow cytometry analysis

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.

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.

Antibodies used for Western blots and IHC

Response of SEMA7A OE cells/tumors to targeted therapies

Characterization of the endocrine resistant phenotype promoted by SEMA7A

H&E and special stains of human breast tissue

Identification of drug concentrations for in vitro analyses

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.

Breast cancer is a leading cause of cancer-associated death in women in the United States, and approximately 70% of all cases are estrogen receptor positive (ER+). While effective ER-targeting endocrine therapy has substantially progressed the successful treatment of primary ER+ tumors, approximately 20% of tumors recur, typically as metastases. Recurrent ER+ tumors consistently develop resistance to endocrine therapies, making them difficult to treat, and contributing to patient death. Therefore, novel molecular targets are needed to treat recurrent ER+ breast cancer. Here, we describe semaphorin 7a (SEMA7A) as a driver of tumor growth, recurrence, and metastasis in ER+ breast cancer. SEMA7A is a signaling protein that is overexpressed in ER+ breast cancer, where it confers significantly decreased patient survival rates for patients on endocrine therapy. We show that SEMA7A is hormonally regulated in ER+ breast cancer; yet, SEMA7A expression does not uniformly decrease in patients treated with endocrine therapies. Instead, endocrine therapy induces one of two outcomes: either 1) decreases SEMA7A and correlates with a good clinical response or 2) increases SEMA7A and correlates with a poor clinical response. We overexpressed SEMA7A in ER+ cell lines and performed in vitro growth assays in the presence of the ER-degrader fulvestrant. We also injected these cells into mammary fat pads of NSG mice, and then treated the animals with fulvestrant. Tumors were measured every three days, then harvested for immunohistochemical analysis. SEMA7A overexpression confers resistance to fulvestrant treatment in vitro and in vivo. Mechanistically, we show that SEMA7A suppresses expression of ER and alters the tumor microenvironment in fulvestrant-treated tumors. These data support SEMA7A as a novel driver of endocrine resistance in ER+ breast cancer. Finally, we identify therapeutic vulnerability of ER+SEMA7A+ tumors and propose that SEMA7A warrants additional investigation in a clinical setting.

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.

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/β1-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.

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 the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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.

This study shows that Rab40b interacts with Cullin5 and that loss of Rab40b–Cullin5 binding affects stress fiber formation and focal adhesion dynamics. Mechanistically, these effects are mediated in part by EPLIN, which is a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation., 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.

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 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.

BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis. Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-11.

Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many of these tumors develop drug resistance and recur within 20 years. Therefore, novel targets are needed to predict for recurrence and to treat recurrent ER+BC. Previous reports describe a tumor-promotional role for Semaphorin 7A (SEMA7A) in ER- disease; yet, the role of SEMA7A in ER+ disease is poorly characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We overexpressed SEMA7A in ER+ BC cells, then used the ER-targeting agents tamoxifen and fulvestrant to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we used proliferation and cell survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to measure how SEMA7A affects tumor growth and metastasis. We also utilized drug resistant cells, which have high endogenous SEMA7A levels, to measure markers of stemness and multi-drug resistance via flow cytometry. Results: We first found that SEMA7A expression correlates with decreased relapse free survival in patients with ER+BC who received endocrine therapy (Kmplotter; p=0.042). We also observe that SEMA7A is hormonally regulated in ER+BC, but its expression does not uniformly decrease with endocrine therapy agents. Instead, long term estrogen deprivation and ER-targeting drug treatments increase SEMA7A expression, likely through the action of other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Further, in ER+ cell lines, overexpression of SEMA7A promotes in vitro growth in the face of estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promotes fulvestrant resistance in the primary tumor and induces lung metastases. Finally, we report that pro-survival signaling is a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies describe that SEMA7A promotes drug resistance in ER+ BC. We propose that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely to respond to endocrine therapies.

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.

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.

BackgroundBreast cancer is a leading cause of cancer-related death for women in the United States. Thus, there a need to investigate novel prognostic markers and therapeutic strategies. Inflammation raises challenges to both treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFkB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, the gene that encodes for cyclooxygenase 2 (COX-2), which is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in inflammation. Here we investigate the effect of Singleminded 2s, a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFkB and COX-2.MethodsWe utilized in vitro reporter assays, immunoblot analyses, qPCR and immunohistochemical analysis to dissect the relationship between NFκB, SIM2s, and COX-2. Furthermore, we utilized COX-2 targeting strategies to determine tumor suppressive activities.ResultsOur results reveal that SIM2s attenuates the activation of a NFκB via luciferase reporter assays. Furthermore, immunostaining of lysates from breast cancer cells over expressing SIM2s showed reduction in various NFκB signaling proteins, whereas knockdown of SIM2 revealed increases in the same NFκB signaling proteins. Additionally, by increasing NFκB translocation to the nucleus in DCIS.COM cells, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increases SIM2s expression. We also found that NFκB/p65 represses SIM2 in via 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 NFkB-mediated suppression of SIM2s. Finally, over expression 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. Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.ConclusionsThese findings provide evidence for a mechanism where SIM2s may represses COX-2 expression to provide an overall better prognosis for breast cancer patients.

Breast cancer (BC) remains the second leading cause of cancer related deaths in women in the US -- mainly due to metastatic disease. Thus, understanding how BC progresses is of the utmost importance to developing new strategies to block metastasis. Young women diagnosed with BC generally have poor prognosis due to increased rates of metastasis. Additionally, women who are within 5 years of most recent child-birth at diagnosis are ∼3 times more likely to develop metastasis than age and stage matched nulliparous women. We define these cases as postpartum breast cancers (PPBC) and propose that the unique biology of the postpartum mammary gland drives tumor progression and metastasis. Semaphorin 7a (SEMA7A) is a unique member of the Semaphorin family as it is the only GPI-anchored member that can be shed into the extracellular environment. Our published results show that SEMA7A expression is associated with decreased overall survival in BC patient cohorts and revealed roles for SEMA7A in breast tumor cell growth, motility, invasion, and tumor associated-lymphangiogenesis all of which are also increased in pre-clinical models of PPBC. However, whether primary SEMA7A driver PPBC progression remains largely unexplored. We utilized a pre-clinical of PPBC with breast cancer cells where we silenced or overexpressed SEMA7A. Our results show that silencing of SEMA7A decreases tumor growth in postpartum hosts while overexpression increases growth in nulliparous hosts to rates closer to those observed in postpartum hosts. Further, we show that SEMA7A effects multiple pro-metastatic tumor attributes such as cell survival, tumor associated COX-2 expression, fibronectin deposition, and fibroblast-mediated collagen deposition in the tumor microenvironment regardless of whether the host is nulliparous or postpartum. Finally, we show that co-expression of SEMA7A/Collagen/COX2/FN mRNA predicts for increased metastasis in breast and ovarian cancer cohorts. These studies suggest SEMA7A as a key mediator of general BC progression and that targeting SEMA7A alone or in combination may open avenues for novel therapeutic strategies to prevent BC progression to metastasis.

We seek to further our understanding of normal mammary gland biology and determine how pregnancy associated changes in the mammary gland contribute to development of breast cancers. Though pregnancy is well-known to provide a protective effect against breast cancer risk, all women who give birth experience a transient increase in breast cancer risk following each pregnancy. The magnitude and length of this elevated risk is largely determined by a woman's age at first birth and for women over 30 at the time of first pregnancy the protective effect likely never occurs. Postpartum breast cancers (PPBC), defined as breast cancers diagnosed within 10 years of last childbirth, are more than twice as likely to become metastatic and result in death. This devastating diagnosis affects ~12,000 women annually. In 2006, for the first time, the number of women having children in their 30s was greater than the number under 25. Thus, PPBC cases are rising. We have shown that tumor cells and normal adjacent mammary epithelial cells (MECs) in PPBC patients exhibit high levels of semaphorin 7a (SEMA7A) protein expression, and SEMA7A expressing tumors are more metastatic. Thus, we believe that understanding the mechanisms underlying SEMA7A signaling in the mammary gland will lead to novel insights into aggressive PPBC. Our recent data reveal that SEMA7A+ MECs are evident during lactation, decreased during the reversible phase of postpartum mammary gland involution, and peak at involution day three, suggesting that SEMA7A promotes survival cell survival during postpartum glandular regression. Consistent with our hypothesis, we reveal that mammary luminal progenitor cells are decreased in SEMA7A knockout mice and SEMA7A promotes cell survival in cultured MECs. Interestingly, a portion of our SEMA7A KO dams exhibit evidence of lactation failure, suggestive of precocious involution. Thus, we propose that SEMA7A activates pro-survival signaling in MECs during lactation and involution. In support of this we show that exogenous SEMA7A activates pro-survival kinase AKT in a β1-integrin dependent manner. Additionally, we show that sustained signaling by SEMA7A leads to cellular transformation in vivo and in vitro and that SEMA7A KO mice crossed to MMTV-PyMT mice exhibit longer latency, ~250 days compared to 50-100 in controls. Since we also observe that SEMA7A levels in blood correlate with SEMA7A levels in breast cancer patient tumors, our results suggest that SEMA7A is a driver of tumorigenesis that could be safely monitored in postpartum women. Citation Format: Taylor R. Rutherford, Traci R. Lyons. Semaphorin 7a promotes cellular transformation via activation of pro-survival signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2672.

Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many tumors become drug resistant and recur within 20 years. Thus, novel targets are needed to treat recurrent ER+BC. Previous reports have described tumor-promotional effects of Semaphorin 7A (SEMA7A) in ER- disease; however, the role of SEMA7A in ER+ disease is not well-characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We measured expression of SEMA7A in drug resistant cells via immunoblotting. We then overexpressed SEMA7A and used ER-targeting drugs (fulvestrant and tamoxifen) to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we utilized cell proliferation and survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to assess how SEMA7A affects primary tumor volume and metastasis. We also measured markers of “stemness” and multi-drug resistance via flow cytometry. Results: We found that SEMA7A expression correlated with decreased relapse free survival in patients who received endocrine therapy for ER+BC (Kmplotter; p=0.042). We also observe that SEMA7A can be hormonally regulated in ER+BC, but expression did not consistently decrease with endocrine therapy. Instead, long term estrogen deprivation and ER-targeting treatments increased SEMA7A expression, likely through signaling via other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Additionally, overexpression of SEMA7A promoted in vitro growth and survival of ER+ cells during estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promoted fulvestrant resistance in the primary tumor and also induced lung metastases. Finally, we identified pro-survival signaling as a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies show that SEMA7A can promote drug resistance in ER+ BC. We suggest that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely be resistant to endocrine therapies. Citation Format: Lyndsey S. Crump, Garhett L. Wyatt, Taylor R. Rutherford, Jennifer K. Richer, Weston W. Porter, Traci R. Lyons. Hormonal regulation of SEMA7A promotes therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1076.

Breast cancer (BC) is the 2nd leading cause of cancer-related death in women. Increased lymphatic vessel density (LVD), lymphovascular invasion (LVI), and lymph node positivity (LN+) are associated with increased metastasis in BC patients. LVD, LVI, and LN+ are promoted by angiogenesis and lymphangiogenesis, which can be induced by tumor-associated macrophages (TAMs). TAMs are implicated in creating a pro-tumor tumor microenvironment (TME) in various cancers. We have shown that semaphorin7A (SEMA7A)—a signaling molecule that activates integrin-mediated signaling cascades—is associated with decreased survival and increased metastasis in BC patients. In mouse models, SEMA7A expression is associated with increased LVD and TAM presence. SEMA7A can also induce expression of lymphatic endothelial cell (LEC)-associated proteins, such as podoplanin (PDPN), on macrophages to polarize them into “PoEMs” (PDPN-expressing macrophages). Furthermore, PoEMs intercalate into lymphatic vessels to form PoEM-LEC chimeric vessels; tumor cells are found within these vessels at the PoEM-LEC junctions. We have also shown that SEMA7A promotes programmed death ligand-1 (PD-L1) expression on TAMs, LECs, PoEMs, and BC cells to inhibit anti-tumor immune responses and promote a pro-tumor TME. In neuronal cells, SEMA7A expression is regulated by steroid hormones in a B1-integrin mediated manner. Given the link between SEMA7A and hormones, we hypothesized that SEMA7A expression in tumors could be induced by estrogen (E2) and that SEMA7A and E2 may cooperate to create a pro-tumor immune TME (iTME). To test this, we orthotopically implanted E0771-DDK (Ctrl) and E0771-DDK-SEMA7A (SEMA7A overexpressing [OE]) cells into female mice with or without E2 supplementation. E0771 cells have recently been characterized as the luminal B subtype—which is ER+ and highly proliferative—and E0771 cells are dependent upon E2 supplementation for growth in vivo. SEMA7A OE tumors without E2 overcame the need for E2 and grew as well as the Ctrl+E2 tumors, but SEMA7A+E2 significantly accelerated tumor growth. Tumors were harvested for flow cytometry (FC) to reveal that E2 increased SEMA7A expression in Ctrl tumors to similar levels observed in the SEMA7A OE and SEMA7A+E2 significantly increased expression of PD-L1 expression on tumor cells. Additionally, SEMA7A+E2 was the only condition to increase the presence of intratumoral LECs; however, E2, SEMA7A, and SEMA7A+E2 all induced PD-L1 expression on LECs. Additionally, SEMA7A and SEMA7A+E2 groups had more TAMs and PoEMs compared to controls, and significantly more of those TAMs/PoEMs were PD-L1+. Lastly, we treated Ctrl and SEMA7A OE tumors (in the presence of E2) with anti-PD1 or anti-PD-L1 therapies and only the SEMA7A OE tumors responded. These results indicate that E2 can induce SEMA7A expression in tumors and that SEMA7A can cooperate with E2 to inhibit anti-tumor immunity. Citation Format: Alan Elder, Lyndsey Crump, Alexander Stoller, Traci Lyons. Semaphorin7A and estrogen work in concert to promote mammary tumor growth and alter the immune tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2791.

Understanding what drives breast tumor progression is of utmost importance for blocking tumor metastasis; we have identified that semaphorin 7a is a potent driver of ductal carcinoma in situ (DCIS) progression. Semaphorin 7a is a glycophosphatidylinositol membrane-anchored protein that promotes attachment and spreading in multiple cell types. Here, we show that increased expression of SEMA7A occurs in a large percentage of breast cancers and is associated with decreased overall and distant metastasis-free survival. In both in vitro and in vivo models, short hairpin-mediated silencing of SEMA7A reveals roles for semaphorin 7a in the promotion of DCIS growth, motility and invasion as well as lymphangiogenesis in the tumor microenvironment. Our studies also uncover a relationship between COX-2 and semaphorin 7a expression and suggest that semaphorin 7a promotes tumor cell invasion on collagen and lymphangiogenesis via activation of β1-integrin receptor. Our results suggest that semaphorin 7a may be novel target for blocking breast tumor progression.

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.

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.

Multiple studies report poor survival rates in postpartum women with breast cancer[1, 2]. A recent study from our group has specifically shown that incidence of recurrence and death from breast cancer is increased approximately 3-fold in postpartum patients compared to nulliparous patients[3]. We define postpartum breast cancers as those diagnosed within 5 years of birth and as a highly metastatic subset of young women’s breast cancer[4]. We have recently shown increased incidence of lymph node involvement in patients diagnosed less than 2 years postpartum and have utilized tissues from our clinical cohort to show increased lymphatic vessel density in stage II postpartum patient samples compared to nulliparous. Using xenograft and isograft murine models we have modeled postpartum breast cancer to show that postpartum tumors are larger[5], exhibit increased lymphatic vessel density, and more frequently spread to mouse lymph node and lung tissues[5] compared to tumors in nulliparous mice. In order to determine whether postpartum tumor cells specifically promote lymphangiogenesis, we utilized postpartum tumor cells ex vivo to show that lymphatic structure formation is promoted via increased postpartum tumor cell secretion of PGE2, which acts on the EP2 receptor on lymphatic endothelial cells. We have also performed expression analyses on postpartum tumor cells isolated from our xenograft model to reveal increased expression of COX-2, VEGF-C, and Semaphorin 7a; all three of these molecules have reported, and possibly interrelated, roles in promotion of lymphangiogenesis[6, 7]. Importantly, both in vivo inhibition and knockdown of COX-2 decrease postpartum tumor associated lymphangiogenesis in pre-clinical models. These results, along with our results indicating that COX-2 inhibition during the postpartum period decreases metastasis in our pre-clinical models, have led us to propose that COX-2 dependent promotion of lymphangiogenesis may facilitate, in part, metastasis of postpartum tumors. 1. Stensheim, H., et al., Cause-specific survival for women diagnosed with cancer during pregnancy or lactation: a registry-based cohort study. J Clin Oncol, 2009. 27(1): p. 45-51. 2. Johansson, A.L., et al., Increased Mortality in Women with Breast Cancer Detected during Pregnancy and Different Periods Postpartum. Cancer Epidemiol Biomarkers Prev, 2011. 20(9): p. 1865-72. 3. Callihan, E.B., et al., Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer. Breast Cancer Res Treat, 2013. 138(2): p. 549-59. 4. Lyons, T.R., P.J. Schedin, and V.F. Borges, Pregnancy and breast cancer: when they collide. J Mammary Gland Biol Neoplasia, 2009. 14(2): p. 87-98. 5. Lyons, T.R., et al., Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med, 2011. 17(9): p. 1109-15. 6. Timoshenko, A.V., et al., COX-2-mediated stimulation of the lymphangiogenic factor VEGF-C in human breast cancer. Br J Cancer, 2006. 94(8): p. 1154-63. 7. Bender, R.J. and F. Mac Gabhann, Expression of VEGF and semaphorin genes define subgroups of triple negative breast cancer. PLoS One, 2013. 8(5): p. e61788. Citation Format: Traci R Lyons, Sarah A Black, Bernard W Futscher, Virginia F Borges, Pepper J Schedin. Promotion of lymphangiogenesis by postpartum breast tumor cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-11.

The limitations of cancer cell lines have led to the development of direct patient derived xenograft (PDX) 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.

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.

Postpartum breast cancers are a highly metastatic subset of young women’s breast cancers defined as breast cancers diagnosed in the postpartum period or within 5 years of last child birth. Women diagnosed with postpartum breast cancer are nearly twice as likely to develop metastasis and to die from breast cancer when compared with nulliparous women. Additionally, epidemiological studies utilizing multiple cohorts also suggest that nearly half of all breast cancers in women aged

Invadopodia formation and extracellular matrix degradation are key events during cancer cell invasion, yet little is known about mechanisms mediating these processes. Here, we report that Rab40b plays a key role in mediating invadopodia function during breast cancer cell invasion. We also identify Tks5 (also known as SH3PXD2A), a known Src kinase substrate, as a new Rab40b effector protein and show that Tks5 functions as a tether that mediates Rab40b-dependent targeting of transport vesicles containing MMP2 and MMP9 to the extending invadopodia. Importantly, we also demonstrate that Rab40b and Tks5 levels are regulated by known tumor suppressor microRNA miR-204. This is the first study that identifies a new Rab40b–Tks5- and miR-204-dependent invadopodia transport pathway that regulates MMP2 and MMP9 secretion, and extracellular matrix remodeling during cancer progression., Highlighted Article: Rab40b plays a key role in mediating invadopodia function during breast cancer cell invasion by binding to Tks5 and functioning as a tether mediating MMP2 and MMP9 targeting to the extending invadopodia.

Key Points Question Is there an increased risk for metastasis of breast cancers that are diagnosed in young women post partum that extends beyond 5 years from the last childbirth, and what association do standard clinical prognostic factors have with metastatic risk in these young women when categorized by parity? Findings In a cohort study of 701 women 45 years or younger with breast cancer, those with stage I or II cancer diagnosed up to 10 years post partum had an increased risk for distant metastasis, with both estrogen receptor–positive and estrogen receptor–negative disease significantly affected. Meaning Postpartum status may be a prognostic indicator in young women with breast cancer and should be routinely identified, as up to 45% of women 45 years or younger with breast cancer fall into this category and could be at increased risk for metastasis., 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., This cohort study evaluates the risk of metastasis following a postpartum breast cancer diagnosis and clinical factors associated with this risk in women 45 years or younger.

Prognosis of young women's breast cancer is influenced by pregnancy. Women diagnosed within 5 years of childbirth have worse prognosis than age-matched nulliparous women or women diagnosed while pregnant. These data indicate that pregnancy-associated breast cancer (PABC) exists as two subtypes, with poor prognosis associated with cases diagnosed in the postpartum setting rather than during pregnancy. We propose that breast involution following pregnancy accounts for the poor prognosis of breast cancers diagnosed within 5 years of childbirth. Characterization of the involuting mammary gland has identified tissue remodeling programs associated with wound healing and inflammatory responses that share similarities with cancer microenvironments known to be tumor promotional. Based on these results, we proposed the Involution Hypothesis, which states that the physiologically normal microenvironment of the postpartum involuting gland is tumor promotional. We estimate a high proportion of young women with breast cancer actually have a “recent” pregnancy as a negative prognostic feature. Specifically, with an upper limit of 40 years of age for reproductive potential and a recent pregnancy defined as within 5 years of diagnosis, an estimated 20,000 cases of breast cancer per year in the U.S. may qualify as postpartum. Targeting the postpartum window with interventions designed to reduce the tumor promotional attributes of involution is highly attractive, as this window of risk is restricted in time and the population is readily identifiable. Testing the Involution Hypothesis has been limited by lack of preclinical models. We describe here a mouse xenograft model of PABC that isolates postpartum mammary gland involution as a driving force for breast cancer progression and metastasis. In this model, human mammary MCFDCIS.com tumor cells injected into the postpartum, involuting mammary gland form more proliferative and invasive tumors than tumor cells injected into the nulliparous gland. The involuting gland is characterized by high levels of radiating collagen fibers organized similar to collagen associated with invasive tumors across species. In our model of postpartum breast cancer, fibrillar collagen is identified as a primary mediator of tumor progression. Mammary tumors arising in the collagen-rich, involuting microenvironment are invasive and have increased levels of COX-2. In vitro, primary tumor cells isolated from PABC tumors are motile and invasive in a COX-2 dependent manner. Evidence that COX-2 induced motility is dependent on collagen signaling is demonstrated with functional blocking antibodies against β1-integrin, 2β1-integrin, and integrin associated protein CD47. These data suggest that treatment targeting COX-2 during postpartum involution may decrease progression of postpartum breast cancer. To test this hypothesis, mice were treated with the NSAID ibuprofen for two weeks coinciding with postpartum mammary gland involution. NSAID treatment was found to reduce tumor size, COX-2 expression, and lung cell seeding in this model. These antitumor effects were observed without blocking apoptosis of secretory mammary epithelial cells, which is required to remodel the gland to a nonsecretory state, and is a requisite for advancing this strategy to clinical trial. The question of whether an NSAID based intervention study could be aimed at recently pregnant women at high risk for breast cancer remains to be determined, but is an extremely desirable objective given that there are more than 6 million pregnancies in the U.S. per year. Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN10-03.

While antiestrogen-based treatments are initially effective for hormone receptor-positive (HR+) breast cancer, these tumors often recur, metastasize, and lose responsiveness to endocrine therapies, emphasizing the need for additional therapeutic targets in HR+ disease. We observe that semaphorin 7a (SEMA7A) expression in HR+ patients in the METABRIC dataset confers significantly decreased survival rates by Kaplan-Meier analysis. Thus, we hypothesize that SEMA7A is a potential biomarker for aggressive disease and/or a novel therapeutic target for recurrent HR+ breast cancer. Utilizing multiple 2D and 3D in vitro approaches with human and mouse HR+ tumor cells, we show that shRNA-mediated knockdown (KD) of SEMA7A results in cell growth defects and reduced invasive potential. Loss of SEMA7A also increases apoptosis in attached and forced suspension culture conditions. Finally, we demonstrate that expression of SEMA7A is regulated by the ovarian hormones estrogen and progesterone in a receptor-dependent mechanism. However, we also observe increased expression of SEMA7A with stabilization of HIF1α, suggesting its expression may be context dependent. Overall, these data support SEMA7A as an important driver of multiple hallmarks of cancer in HR+ breast cancer, including cell growth, invasion, and resisting death. As SEMA7A expression is low in normal breast cells, it may be a novel therapeutic target with low toxic side effects. Citation Format: Lyndsey Crump, Alan Elder, Sarah Tarullo, Paris Franz, Traci Lyons. Investigating semaphorin 7a in hormone receptor-positive breast cancer progression [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B54.

Despite new targeted therapies and improved screening, breast cancer is the second leading cause of cancer-related death of women in the United States. This emphasizes the need to better understand the molecular pathways contributing to breast cancer progression and metastasis and the need for identification of novel therapeutic targets. This is especially true for triple-negative breast cancers (TNBCs), which in comparison to hormone receptor-positive (ER+/PR+) and HER2+ subtypes lack a targeted therapy. Without targeted therapy TNBC patients have limited options for treatment, resulting in decreased overall survival. Our lab identified semaphorin 7a (SEMA7A) as a novel protein that promotes TNBC progression. SEMA7A is a member of the semaphorin family, a group of proteins originally described for their developmental roles in axon guidance. SEMA7A is a unique member of the semaphorin family as it is the only GPI-linked semaphorin and can be shed into the extracellular space. Since breast cancer patients mainly die from metastatic disease, we aim to uncover the role of SEMA7A in TNBC cell invasion and metastasis. To study the role SEMA7A may have in promoting cellular invasion, we utilized an shRNA-mediated knockdown of SEMA7A in human TNBC cell lines. Our results show that SEMA7A plays an important role in many steps of the metastatic cascade, with shSEMA7A cells showing increased cell death in both attached and force suspension conditions (anoikis), reduced mesenchymal phenotypes, and less invasion in 3D matrices. We also observed decreased growth and invasion of shSEMA7A tumors in vivo and decreased synthesis of fibronectin and matrix-metalloproteinase 2 ex vivo from shSEMA7A tumors. Taken together, these results show that SEMA7A is a critical molecule for cellular invasion by modulating not only intrinsic cellular pathways, but also the tumor microenvironment. Citation Format: Sarah Elizabeth Tarullo, Alan Elder, Ryan Hill, Kirk Hansen, Traci Lyons. Investigating the role of semaphorin 7a in triple-negative breast cancer cell invasion [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B55.

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.