The Impact of Mesothelin in the Ovarian Cancer Tumor Microenvironment

Ovarian cancer is the deadliest gynecological disease among U.S. women. Poor 5-year survival rates (<30%) are due to presentation of most women at diagnosis with advanced stage disease with widely disseminated intraperitoneal metastasis. However, when diagnosed before metastatic propagation the overall 5-year survival rate is >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. In this review, the involvement of mesothelin (MSLN) in the tumor microenvironment is discussed. MSLN, a 40kDa glycoprotein that is overexpressed 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 MSLN 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. Although the cancer-specific expression of MSLN makes it a potential therapeutic target, more studies are needed to validate the role of MSLN in tumor metastasis

Gonadotropins Regulate Oncogenic Pathways in Ovarian Surface Epithelium in a Three-Dimensional Model

Ovarian cancer is the most lethal gynecological malignancy among American women, killing 70% of women diagnosed due to the lack of signs and symptoms leading to late diagnosis. Epithelial ovarian carcinomas are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation associated with ovulation, and the gonadotropins that regulate ovulation have been suggested to play a role in ovarian surface transformation and cancer progression. The elevated levels of gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), during ovulation, menopause, and premature ovarian failure have been hypothesized to play a role in the initiation and progression of ovarian cancer. Aspects of ovarian surface repair following ovulation include proliferation, migration, and surface regeneration. In order to study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 day of culture. Organ cultures were propagated in medium supplemented with bovine serum albumin (BSA) or fetal bovine serum (FBS) to determine optimal growth conditions. OSE from BSA cultured organs proliferated significantly more than controls until day 4 while organs cultured in FBS had significantly more surface area encapsulated by OSE. The role of the gonadotropins in normal OSE is not well characterized; however, previous in vivo studies demonstrated that the gonadotropins increased OSE proliferation but in vitro studies were inconclusive because the OSE cells studied were usually immortalized and underwent morphological changes. Using a three-dimensional (3D) organ culture system, the role of gonadotropins in normal OSE in the absence of ovulation was investigated to test the hypothesis that the gonadotropins increase OSE proliferation by activating oncogenic pathways that may lead to induction of ovarian cancer. The 3D ovarian organ culture confirmed previous in vivo studies that demonstrated FSH and LH promote proliferation of the OSE. Transcriptional array analyses demonstrated the gonadotropins activated pathways that control cell cycle, angiogenesis and metastasis. Akt and epidermal growth factor receptor (EGFR) signaling were integral to gonadotropin induced OSE proliferation as demonstrated by abrogation by specific chemical inhibitors of these pathways. Western blots confirmed that Akt expression is not only enhanced, but Akt is also phosphorylated from FSH and LH treatment. However, inhibition of Akt signaling did not completely suppress Akt expression in LH treated ovaries, likely due to upregulation of both Akt1 and Akt2. Soft agar transformation experiments indicated menopausal concentrations of LH initiated OSE transformation. When hydrogen peroxide was used to initiate transformation to mimic oxidative stress from ovulation, FSH progressed OSE colony formation at menopausal concentrations. Although investigating the initiation and progression of the disease is crucial, alternative options for drug therapy are just as important. The current drug therapy used to treat ovarian cancer is paclitaxel/carboplatin, but most women develop drug resistance and recurrence of the disease, necessitating alternative strategies for treatment. A possible molecular target for cancer therapy is glycogen synthase kinase 3β (GSK3β), a downstream kinase in the Wnt signaling pathway that is overexpressed in serous ovarian cancer. Novel maleimide-based GSK3β inhibitors (GSK3βi) were synthesized, selected, and tested in vitro using SKOV3 and OVCA432 serous ovarian cancer cell lines. From a panel of 10 inhibitors, GSK3βi 9ING41 was found to be the most effective in vitro. 9ING41 induced apoptosis as indicated by 4′6-diamidino-2-phenylindole-positive nuclear condensation (DAPI), poly (ADP-ribose) polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The mechanism for apoptosis was through caspase-3 cleavage. GSK3βi upregulated phosphorylation of the inhibitory serine residue of GSK3β in OVCA432 and SKOV3 cell lines and also inhibited phosphorylation of the downstream target glycogen synthase. A xenograft study using SKOV3 cells demonstrated that tumor growth was hindered by 9ING41 in vivo. Therefore, GSK3β inhibitors alone or in combination with existing drugs may hinder the growth of serous ovarian cancers. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer. Also, the 3D ovarian organ culture identified key cancer pathways regulated by gonadotropins and revealed how these hormones might contribute to OSE transformation by increasing proliferation, initiating, and progressing colony formation stimulated by oxidative stress. By testing and validating drug targets downstream of FSH and LH signaling, such as GSK3, perhaps ovarian cancers can not only be stifled at an earlier stage but be treated with more targeted therapies

Gonadotropins Activate Oncogenic Pathways to Enhance Proliferation in Normal Mouse Ovarian Surface Epithelium

Ovarian cancer is the most lethal gynecological malignancy affecting American women. The gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), have been implicated as growth factors in ovarian cancer. In the present study, pathways activated by FSH and LH in normal ovarian surface epithelium (OSE) grown in their microenvironment were investigated. Gonadotropins increased proliferation in both three-dimensional (3D) ovarian organ culture and in a two-dimensional (2D) normal mouse cell line. A mouse cancer pathway qPCR array using mRNA collected from 3D organ cultures identified Akt as a transcriptionally upregulated target following stimulation with FSH, LH and the combination of FSH and LH. Activation of additional pathways, such as Birc5, Cdk2, Cdk4, and Cdkn2a identified in the 3D organ cultures, were validated by western blot using the 2D cell line. Akt and epidermal growth factor receptor (EGFR) inhibitors blocked gonadotropin-induced cell proliferation in 3D organ and 2D cell culture. OSE isolated from 3D organ cultures stimulated with LH or hydrogen peroxide initiated growth in soft agar. Hydrogen peroxide stimulated colonies were further enhanced when supplemented with FSH. LH colony formation and FSH promotion were blocked by Akt and EGFR inhibitors. These data suggest that the gonadotropins stimulate some of the same proliferative pathways in normal OSE that are activated in ovarian cancers

Maternal obesity driven changes in collagen linearity of breast extracellular matrix induces invasive mammary epithelial cell phenotype

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.</p

Another wrinkle with age: Aged collagen and intra‐peritoneal metastasis of ovarian cancer

Abstract Background Age is the most significant risk factor for ovarian cancer (OvCa), the deadliest gynecologic malignancy. Metastasizing OvCa cells adhere to the omentum, a peritoneal structure rich in collagen, adipocytes, and immune cells. Ultrastructural changes in the omentum and the omental collagen matrix with aging have not been evaluated. Aim The aim of this study was to test the hypothesis that age‐related changes in collagen in the ovarian tumor microenvironment promote OvCa metastatic success in the aged host. Methods/Results Young (3–6 months) and aged mice (20–23 months) were used to study the role of aging in metastatic success. Intra‐peritoneal (IP) injection of ID8Trp53–/– OvCa cells showed enhanced IP dissemination in aged versus young mice. In vitro assays using purified collagen demonstrated reduced collagenolysis of aged fibers, as visualized using scanning electron microscopy (SEM) and quantified with a hydroxyproline release assay. Omental tumors in young and aged mice showed similar collagen deposition; however enhanced intra‐tumoral collagen remodeling was seen in aged mice probed with a biotinylated collagen hybridizing peptide (CHP). In contrast, second harmonic generation (SHG) microscopy showed significant differences in collagen fiber structure and organization in omental tissue, and SEM demonstrated enhanced omental fenestration in aged omenta. Combined SHG and Alexa Fluor‐CHP microscopy in vivo demonstrated that peri‐tumoral collagen was remodeled more extensively in young mice. This collagen population represents truly aged host collagen, in contrast to intra‐tumoral collagen that is newly synthesized, likely by cancer‐associated fibroblasts. Conclusions Our results demonstrate that tumors in an aged host can grow with minimal collagen remodeling, while tumors in the young host must remodel peri‐tumoral collagen to enable effective proliferation, providing a mechanism whereby age‐induced ultrastructural changes in collagen and collagen‐rich omenta establish a permissive premetastatic niche contributing to enhanced OvCa metastatic success in the aged host

Advanced Glycation End Products as a Potential Target for Restructuring the Ovarian Cancer Microenvironment: A Pilot Study

Ovarian cancer is the sixth leading cause of cancer-related death in women, and both occurrence and mortality are increased in women over the age of 60. There are documented age-related changes in the ovarian cancer microenvironment that have been shown to create a permissive metastatic niche, including the formation of advanced glycation end products, or AGEs, that form crosslinks between collagen molecules. Small molecules that disrupt AGEs, known as AGE breakers, have been examined in other diseases, but their efficacy in ovarian cancer has not been evaluated. The goal of this pilot study is to target age-related changes in the tumor microenvironment with the long-term aim of improving response to therapy in older patients. Here, we show that AGE breakers have the potential to change the omental collagen structure and modulate the peritoneal immune landscape, suggesting a potential use for AGE breakers in the treatment of ovarian cancer

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

Three-Dimensional Ovarian Organ Culture as a Tool to Study Normal Ovarian Surface Epithelial Wound Repair

Ovarian cancers are primarily derived from a single layer of epithelial cells surrounding the ovary, the ovarian surface epithelium (OSE). Ovarian surface proliferation is associated with ovulation and has been suggested to play a role in ovarian surface transformation and cancer progression. Aspects of ovarian surface repair after ovulation include proliferation, migration, and surface regeneration. To study ovarian surface repair, an organ culture system was developed that supports the proliferation, encapsulation, and repair of an artificially wounded surface. Wounded mouse ovaries embedded into an alginate hydrogel matrix have normal OSE cells as demonstrated by expression of cytokeratin 8, vimentin, N-cadherin, and a lack of E-cadherin. Normal OSE cells began proliferating and migrating around wounded surfaces after 1 d of culture. Organ cultures were propagated in medium supplemented with BSA and fetal bovine serum to determine optimal growth conditions. BSA cultured organs had OSE that proliferated significantly more than controls until d 4, whereas fetal bovine serum cultured organs had significantly more surface area encapsulated by OSE. Overall, a three-dimensional ovarian organ culture supports the growth of normal OSE in response to artificial wounding and provides a novel system for investigating wound repair as it relates to the possible role of ovulation and ovarian cancer

Host obesity alters the ovarian tumor immune microenvironment and impacts response to standard of care chemotherapy

Background: The majority of women with epithelial ovarian cancer (OvCa) are diagnosed with metastatic disease, resulting in a poor 5-year survival of 31%. Obesity is a recognized non-infectious pandemic that increases OvCa incidence, enhances metastatic success and reduces survival. We have previously demonstrated a link between obesity and OvCa metastatic success in a diet-induced obesity mouse model wherein a significantly enhanced tumor burden was associated with a decreased M1/M2 tumor-associated macrophage ratio (Liu Y et al. Can, Res. 2015; 75:5046-57).Methods: The objective of this study was to use pre-clinical murine models of diet-induced obesity to evaluate the effect of a high fat diet (HFD) on response to standard of care chemotherapy and to assess obesity-associated changes in the tumor microenvironment. Archived tumor tissues from ovarian cancer patients of defined body mass index (BMI) were also evaluated using multiplexed immunofluorescence analysis of immune markers.Results: We observed a significantly diminished response to standard of care paclitaxel/carboplatin chemotherapy in HFD mice relative to low fat diet (LFD) controls. A corresponding decrease in the M1/M2 macrophage ratio and enhanced tumor fibrosis were observed both in murine DIO studies and in human tumors from women with BMI > 30.Conclusions: Our data suggest that the reported negative impact of obesity on OvCa patient survival may be due in part to the effect of the altered M1/M2 tumor-associated macrophage ratio and enhanced fibrosis on chemosensitivity. These data demonstrate a contribution of host obesity to ovarian tumor progression and therapeutic response and support future combination strategies targeting macrophage polarization and/or fibrosis in the obese host.</p