Elizabeth Loughran

Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration.

IntroductionThe Rho GTPase Cdc42 is overexpressed and hyperactivated in breast tumors compared to normal breast tissue. Cdc42 regulates key processes that are critical for mammary gland morphogenesis and become disrupted during the development, progression, and metastasis of breast cancer. However, the contribution of Cdc42 to normal and neoplastic mammary gland development in vivo remains poorly understood. We were therefore interested in investigating the effects of Cdc42 overexpression on mammary gland morphogenesis as a first step toward understanding how its overexpression may contribute to mammary tumorigenesis.MethodsWe developed a tetracycline-regulatable Cdc42 overexpression mouse model in which Cdc42 can be inducibly overexpressed in the developing mammary gland. The effects of Cdc42 overexpression during postnatal mammary gland development were investigated using in vivo and in vitro approaches, including morphometric analysis of wholemounted mammary glands, quantification of histological markers, and primary mammary epithelial cell (MEC) functional and biochemical assays.ResultsAnalysis of Cdc42-overexpressing mammary glands revealed abnormal terminal end bud (TEB) morphologies, characterized by hyperbudding and trifurcation, and increased side branching within the ductal tree. Quantification of markers of proliferation and apoptosis suggested that these phenotypes were not due to increased cell proliferation or survival. Rather, Cdc42 overexpressing MECs were more migratory and contractile and formed dysmorphic, invasive acini in three-dimensional cultures. Cdc42 and RhoA activities, phosphorylated myosin light chain, and MAPK signaling, which contribute to migration and invasion, were markedly elevated in Cdc42 overexpressing MECs. Interestingly, Cdc42 overexpressing mammary glands displayed several features associated with altered epithelial-stromal interactions, which are known to regulate branching morphogenesis. These included increased stromal thickness and collagen deposition, and stromal cells isolated from Cdc42 overexpressing mammary glands exhibited elevated mRNA expression of extracellular matrix proteins and remodeling enzymes.ConclusionsThese data suggest that Cdc42 overexpression disrupts mammary gland branching morphogenesis by altering Rho GTPase and MAPK signaling, leading to increased MEC contractility and migration in association with stromal alterations. Our studies provide insight into how aberrant Cdc42 expression may contribute to mammary tumorigenesis.

Heterogeneous Cadherin Expression and Multicellular Aggregate Dynamics in Ovarian Cancer Dissemination

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: “mixed cadherin,” in which distinct cells within the same tumor express either E- or Ncad, and “hybrid cadherin,” wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of “mixed cadherin” MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the “hybrid cadherin” Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success.

Aging Increases Susceptibility to Ovarian Cancer Metastasis in Murine Allograft Models and Alters Immune Composition of Peritoneal Adipose Tissue

Ovarian cancer, the most deadly gynecological malignancy in U.S. women, metastasizes uniquely, spreading through the peritoneal cavity and often generating widespread metastatic sites before diagnosis. The vast majority of ovarian cancer cases occur in women over 40 and the median age at diagnosis is 63. Additionally, elderly women receive poorer prognoses when diagnosed with ovarian cancer. Despite age being a significant risk factor for the development of this cancer, there are little published data which address the impact of aging on ovarian cancer metastasis. Here we report that the aged host is more susceptible to metastatic success using two murine syngeneic allograft models of ovarian cancer metastasis. This age-related increase in metastatic tumor burden corresponds with an increase in tumor infiltrating lymphocytes (TILs) in tumor-bearing mice and alteration of B cell-related pathways in gonadal adipose tissue. Based on this work, further studies elucidating the status of B cell TILs in mouse models of metastasis and human tumors in the context of aging are warranted.

Quantitative proteomic analysis of murine white adipose tissue for peritoneal cancer metastasis

AbstractCancer metastasis risk increases in older individuals, but the mechanisms for this risk increase are unclear. Many peritoneal cancers, including ovarian cancer, preferentially metastasize to peritoneal fat depots. However, there is a dearth of studies exploring aged peritoneal adipose tissue in the context of cancer. Because adipose tissue produces signals which influence several diseases including cancer, proteomics of adipose tissue in aged and young mice may provide insight into metastatic mechanisms. We analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. We identified 2308 protein groups and quantified 2167 groups, among which several protein groups showed twofold or greater abundance differences between the aged and young cohorts. Cancer-related gene products previously identified as significant in another age-related study were found altered in this study. Several gene products known to suppress proliferation and cellular invasion were found downregulated in the aged cohort, including R-Ras, Arid1a, and heat shock protein β1. In addition, multiple protein groups were identified within single cohorts, including the proteins Cd11a, Stat3, and Ptk2b. These data suggest that adipose tissue is a strong candidate for analysis to identify possible contributors to cancer metastasis in older subjects. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged. Graphical abstractWe analyzed mesenteric, omental, and uterine adipose tissue groups from the peritoneal cavities of young and aged C57BL/6J mouse cohorts with a low-fraction SDS-PAGE gelLC-MS/MS method. These fat depots are preferential sites for many peritoneal cancers. The results of this study, the first of its kind using uterine adipose tissue, contribute to the understanding of the role of adipose tissue in age-related alteration of oncogenic pathways, which may help elucidate the mechanisms of increased metastatic tumor burden in the aged.

Multiparity activates interferon pathways in peritoneal adipose tissue and decreases susceptibility to ovarian cancer metastasis in a murine allograft model

Ovarian cancer is the fifth leading cause of cancer deaths in U.S. women and the deadliest gynecologic malignancy. This lethality is largely due to the fact that most cases are diagnosed at metastatic stages of the disease when the prognosis is poor. Epidemiologic studies consistently demonstrate that parous women have a reduced risk of developing ovarian cancer, with a greater number of births affording greater protection; however little is known about the impact of parity on ovarian cancer metastasis. Here we report that multiparous mice are less susceptible to ovarian cancer metastasis in an age-matched syngeneic murine allograft model. Interferon pathways were found to be upregulated in healthy adipose tissue of multiparous mice, suggesting a possible mechanism for the multiparous-related protective effect against metastasis. This protective effect was found to be lost with age. Based on this work, future studies exploring therapeutic strategies which harness the multiparity-associated protective effect demonstrated here are warranted.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.

Abstract TMEM-025: IMPACT OF MESOTHELIN EXPRESSION ON THE METASTATIC SUCCESS OF OVARIAN CANCER

Ovarian cancer is the most lethal gynecological cancer in U.S. women. Poor 5-year survival rates ( 90%. Metastasizing tumor cells grow rapidly and aggressively attach to the mesothelium of all organs within the peritoneal cavity, including the parietal peritoneum and the omentum, producing secondary lesions. Mesothelin (MSLN), a 40kDa glycoprotein that is over expressed in many cancers including ovarian and mesotheliomas is suggested to play a role in cell survival, proliferation, tumor progression and adherence. However, the biological function of mesothelin is not fully understood as MSLN knockout mice do not present with an abnormal phenotype. Conversely, MSLN has been shown to bind to the ovarian cancer antigen, CA-125, and thought to play a role in the peritoneal diffusion of ovarian tumor cells. Taking into consideration the potential importance of MSLN/CA-125 binding in ovarian tumor metastasis within the peritoneum, MSLN wild type (WT) and knockout (KO) mice were used to explore the role of mesothelin on the susceptibility of ovarian tumor cells to adhere to the mesothelium of the organs in the peritoneal cavity. An ex vivo peritoneal assay, using CA-125 positive human ovarian tumor cells OVCAR8-GFP and peritoneal explants from MSLN WT and KO mice demonstrated a decrease in OVCAR8-GFP cell adhesion to peritoneal tissues from MSLN KO mice compared to MSLN WT mice. Furthermore, allograft tumor studies using MSLN WT and KO mice injected intraperitoneally with fluorescently-tagged syngeneic murine ovarian cancer cells (ID8-RFP) was performed. Disease progression was evaluated post injection by fluorescent in vivo imaging prior to end point dissection (~8 weeks). Abdominal organs were dissected, imaged ex vivo and organ-specific tumor burden was quantified by tumor area. Tumor burden was significantly decreased in the liver and omentum of MSLN KO mice compared to MSLN WT mice. Together, the results demonstrate a loss of mesothelial cell-ovarian tumor cell adhesion in the omentum and peritoneum of mice that do not express MSLN. Citation Format: Tyvette Hilliard, Kyle Iwamoto, Elizabeth Loughran, Marwa Asem, Yueying Liu, Jing Yang, Laura Tarwater, Yuliya Klymenko, Jeff Johnson, Zonggao Shi, and M. Sharon Stack. IMPACT OF MESOTHELIN EXPRESSION ON THE METASTATIC SUCCESS OF OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-025.

Abstract TMEM-029: AGING INCREASES SUSCEPTIBILITY TO OVARIAN CANCER METASTASIS IN A MURINE ALLOGRAFT MODEL

Ovarian cancer (OvCa) is the leading gynecological malignancy in women in the United States. OvCa metastasizes uniquely, spreading through the peritoneal cavity and generating widespread metastatic sites. The vast majority of OvCa cases occur in women over 40 and the median age at diagnosis is 63 (SEER). Despite age being a significant risk factor for the development of OvCa, there is a paucity of studies addressing the role of aging in OvCa metastasis. To our knowledge, there are no reports utilizing old mice to investigate the effects of age on metastasis in vivo . We designed a study using a C57BL/6 model of aging where young (Y) mice are 3-6 months of age and aged (A) mice are 20-23 months of age, corresponding to young (20-30 years) and aged (60-67 years) humans. Using the C57BL/6 syngeneic ID8 mouse ovarian surface epithelial cell line, we tested the effect of aging on metastatic success in vivo . An allograft study was carried out with Y and A mice that were intraperitoneally injected with 3.7x10 6 ID8 RFP-tagged cells. The mice were imaged once a week starting at 4.5 weeks post injection and were sacrificed for dissection at 8 weeks post injection. Live imaging suggested OvCa metastasis was more efficient in the aged animals than in the young animals. After dissection, the abdominal organs were imaged ex vivo and tumor burden was quantified. The aged mice displayed heavier tumor burden in the gonadal fat compared to the young. Interestingly, no difference in metastasis to the omentum was detected. To investigate why gonadal fat is more receptive to metastasis in the aged animals, periovarian adipose from 4 young and 4 aged healthy non-tumor bearing mice was isolated for RNAseq analysis. Several immune pathways involving B cells were found to be significantly upregulated in the RNA from aged animals. Studies will be conducted to elucidate the status of B cells in aging periovarian adipose, including immunohistochemistry for CD45 and other B cell markers upregulated in the RNAseq dataset. Citation Format: Elizabeth Loughran, Annemarie Leonard, Ryan Phan, Laura Tarwater, Tyvette Hilliard, Marwa Asem, Yueying Liu, Jing Yang, Yuliya Klymenko, Jeff Johnson, Zonggao Shi, Matthew Leevy, Matthew Ravosa and M. Sharon Stack. AGING INCREASES SUSCEPTIBILITY TO OVARIAN CANCER METASTASIS IN A MURINE ALLOGRAFT MODEL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-029.

Abstract B67: The impact of parity on the metastatic success of ovarian cancer.

Ovarian cancer is the most fatal gynecological cancer. Epithelial ovarian cancer (OvCa), the most common subtype, usually goes undetected until metastatic and often fatal stages of the disease. OvCa follows a unique form of metastasis, spreading through the peritoneal cavity and forming metastatic sites on the peritoneum. Epidemiologic data suggest that child-bearing, or parity, reduces a woman9s risk of developing ovarian cancer, with more births providing greater protection. Despite the association of parity with a decreased incidence of ovarian cancer, very few studies have explored the relationship between parity and metastatic success. A recent study compared metastatic success to the omentum in 12-month-old C57Bl/6 retired breeders and 5-month-old virgin mice, reporting that parous mice are less susceptible to metastasis due to the parity-associated differences in the immune compositional profile in the omental fat band (Cohen et al. 2013). This tumor study compared mice of different ages and did not report specific numbers of pregnancies. To further investigate the role of parity number in OvCa metastasis, we designed a study that controls for age and compares mice with specific parity number. Three age-matched C57Bl/6 groups were evaluated: nulliparous (V), parous 1 (P1), and parous 3 (P3) mice. We tested the effect of parity on metastatic success in vivo with an allograft study using the C57Bl/6 syngeneic ID8 mouse ovarian surface epithelial cell line. ID8 ovarian cancer cells (106) were injected into the peritoneal cavity of V, P1 and P3 mice. In contrast to the results of Cohen at al. that utilized a different syngeneic ovarian cancer cell line, we found no significant difference in metastasis to the omentum in the parous animals, but significantly reduced metastasis to the fat-enveloped ovaries and visceral fat pads in the P3 mice. This suggests that the visceral fat adjacent to the uterus and ovaries in multi-parous animals is a unique environment, resilient to metastasis. Factors in fat tissue responsible for this phenomenon are being investigated. Citation Format: Elizabeth A. Loughran, Ryan Phan, Annemarie K. Leonard, Laura Tarwater, Marwa Asem, Yuliya Klymenko, Yueying Liu, Jing Yang, Jeff Johnson, Matthew Ravosa, M. Sharon Stack. The impact of parity on the metastatic success of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B67.