Androgens, although traditionally thought to be male sex steroids, play important roles in female reproduction, both in healthy and pathological states. This mini-review focuses on recent advances in our knowledge of the role of androgens in the ovary. Androgen receptor (AR) is expressed in oocytes, granulosa cells, and theca cells, and is temporally regulated during follicular development. Mouse knockout studies have shown that AR expression in granulosa cells is critical for normal follicular development and subsequent ovulation. In addition, androgens are involved in regulating dynamic changes in ovarian steroidogenesis that are critical for normal cycling. Androgen effects on follicle development have been incorporated into clinical practice in women with diminished ovarian reserve, albeit with limited success in available literature. At the other extreme, androgen excess leads to disordered follicle development and anovulatory infertility known as polycystic ovary syndrome (PCOS), with studies suggesting that theca cell AR may mediate many of these negative effects. Finally, both prenatal and postnatal animal models of androgen excess have been developed and are being used to study the pathophysiology of PCOS both within the ovary and with regard to overall metabolic health. Taken together, current scientific consensus is that a careful balance of androgen activity in the ovary is necessary for reproductive health in women.
Chronic inflammation promotes progression of many cancers, with circulating myeloid-derived suppressor cell (MDSC) levels correlating with poor prognosis. Here we examine effects of MDSCs on lymphangioleiomyomatosis (LAM), a rare disease occurring almost exclusively in women whereby estrogen-sensitive metastatic TSC2-null tumors grow throughout the lungs, markedly reducing pulmonary function. The LAM cell origin remains unknown; however, previous work demonstrated that Tsc2 inactivation in the mouse uterus induced estrogen-dependent myometrial tumors with nearly all features of LAM. Half of these animals developed metastatic myometrial tumors in the lungs, suggesting that LAM cells might originate from the myometrium, possibly explaining its overwhelming female prevalence and estrogen-sensitivity. Here we report that MDSC levels, and in particular granulocytic myeloid cell levels, are elevated in the periphery and in tumors of uterine-specific Tsc2-null mice. Importantly, MDSC depletion or inhibition of their recruitment impairs myometrial tumor growth. RNA and protein analysis of Tsc2-null myometrial tumors and xenografts demonstrate high expression and activity of the serine protease neutrophil elastase (NE), with selective qPCR studies indicating a stromal origin of the NE. Notably, treatment with sivelestat, a known NE inhibitor already approved for human use in some countries, reduces tumor growth similar to MDSC depletion. Furthermore, NE promotes Tsc2-null tumor cell growth, migration, and invasion in vitro. Finally, NE-expressing myeloid cells are present throughout the lungs of LAM patients but not controls. These data suggest that NE derived from granulocytic myeloid cells might directly promote LAM tumor cell progression and could be a novel therapeutic target for LAM.
Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease caused by smooth muscle cell-like tumors containing tuberous sclerosis (TSC) gene mutations and found almost exclusively in females. Patient studies suggest LAM progression is estrogen-dependent, an observation supported by in vivo mouse models. However, in vitro data using TSC-null cells lines demonstrate modest estradiol responses, suggesting estradiol effects in vivo may involve pathways independent of direct tumor stimulation. We previously reported tumor-dependent neutrophil expansion and promotion of TSC2-null tumor growth in an estradiol-sensitive LAM mouse model. We therefore hypothesized that estradiol stimulates tumor growth in part by promoting neutrophil production. Here we report that estradiol-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that estradiol induces granulopoiesis via ERα in male and female bone marrow cultures. With our novel TSC2-null mouse myometrial cell line, we show that factors released from these cells drive estradiol-sensitive neutrophil production. Lastly, we analyzed single-cell RNA sequencing data from LAM patients and demonstrate the presence of tumor-activated neutrophils. Our data suggest a powerful positive feedback loop whereby estradiol and tumor factors induce neutrophil expansion, which in turn intensifies tumor growth and production of neutrophil-stimulating factors, resulting in continued TSC2-null tumor growth.
Predominately affecting women of reproductive age, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. We have previously reported that estrogen is required to maintain LAM-like tumor progression in a uterine-specific Tsc2-knockout murine model. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol (E2) in vitro, suggesting that estradiol may act elsewhere to promote LAM progression. We hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting polymorphonuclear cell (PMN) production in the bone marrow and actions in the tumor microenvironment. Here, using bone marrow cultures, we demonstrate that estradiol is a potent inducer of PMN production. This effect occurs equally with both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ER_ knockout mice, we showed that ER_ is necessary for promoting a PMN fate for myeloid progenitors. While we see that the migratory and invasive capacities of TSC2-null cell lines are not augmented when stimulated with E2 directly, evidence shows estrogen promotes the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. These data provide insight into the robust effect of in vivo estrogen stimulation as estradiol may be a dual effector in LAM tumor progression and great target for anti-LAM therapeutic strategy.
Affecting almost exclusively women, lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by slowly growing, estrogen-sensitive metastatic smooth muscle cell-like adenomas that result in cystic lung changes and loss of pulmonary function. LAM tumors are caused by mutations in either TSC1 or TSC2 genes that induces defective inhibition of the mTORC1 pathway, leading to increased mTORC1 activity and augmented cell proliferation. We have previously reported that estrogen ablation in our uterine-specific Tsc2 knockout mouse, which grows tumors with characteristic LAM features and lung colonization potential, effects notable regression of tumors. Thus, estrogen is required for to maintain heightened mTORC1 activity and LAM-like tumor progression. Interestingly, the observed estrogen sensitivity in vivo is more markedly pronounced than that of our estrogen receptor-positive TSC2-null cells when stimulated with estradiol in vitro, suggesting that estradiol may act elsewhere—in mTORC1 independent manner—in vivo to promote LAM progression. Flow cytometry revealed large numbers of Ly-6Cint Ly-6Ghigh myeloid cells—polymorphonuclear cells or PMNs—in the blood and myometrial tumors of our uterine-specific Tsc2-null mice. Accordingly, we found that Tsc2-null tumors required PMNs for normal disease progression, as Gr-1 (Ly-6C/Ly-6G) depletion or inhibition of PMN recruitment reduced tumor growth. Therefore, we hypothesized that, in addition to direct effects of estrogen on tumor cells, estrogen might also stimulate tumor growth by promoting PMN production in the bone marrow and actions in the tumor microenvironment. Using bone marrow cultures, we found that estradiol is indeed a potent inducer of PMN production. This effect occurs equally in both male and female bone marrow. Employing both pharmacologic agents and bone marrow from ERα; knockout mice, we showed that ERα; is necessary for promoting a PMN fate for myeloid progenitors. Additionally, we have evidence implicating estrogen in the pro-tumorigenic function of PMNs co-cultured with TSC2-null cell lines. Overall, these data suggest that estradiol maybe facilitating crosstalk in LAM tumors, directly stimulating tumor cells while also promoting the production and actions of PMNs, which in turn promote tumor growth.
Lymphangioleiomyomatosis (LAM) is a cystic lung disease found almost exclusively in genetic females and caused by small clusters of smooth muscle cell tumors containing mutations in one of the two tuberous sclerosis genes (TSC1 or TSC2). Significant advances over the past 2-3 decades have allowed researchers and clinicians to more clearly understand the pathophysiology of LAM, and therefore better diagnose and treat patients with this disease. Despite substantial progress, only one proven treatment for LAM is used in practice: mTORC1 inhibition with medications such as sirolimus. While mTORC1 inhibition effectively slows LAM progression in many patients, it is not curative, is not effective in all patients, and can be associated with significant side effects. Furthermore, the presence of established and accurate biomarkers to follow LAM progression is limited. That said, discovering additional diagnostic and treatment options for LAM is paramount. This review will describe recent advances in LAM research, centering on the origin and nature of the LAM cell, the role of estrogen in LAM progression, the significance of melanocytic marker expression in LAM cells, and potential roles of the microenvironment in promoting LAM tumor growth. By appreciating these processes in more detail, researchers and caregivers may be afforded novel approaches to aid in the treatment of patients with LAM.
Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women. LAM is characterized by the hyperproliferation of smooth muscle cells creating small tumors throughout the lungs, resulting in the formation of large cysts that replace normal alveolar space. Growth of these tumors and progression of the cyst development leads to loss of pulmonary function, and sometimes subsequent lung transplantation. LAM tumor cells contain mutations in one of the tuberous sclerosis genes (TSC1 or TSC2), leading to activation of the mTORC1 pathway. In fact, mTOR inhibitors are commonly used to treat LAM; however, these drugs are not always effective and have significant side effects, suggesting the need for new therapeutic targets. Additionally, tumors recur even after lung transplantation and LAM cells are found in circulating body fluids, suggesting a metastatic nature of LAM, and a question of the origin of the LAM cell. Due to LAM’s estrogen sensitivity, female specificity, and metastatic nature, we previously proposed that LAM cells originate from the uterine myometrium. We therefore designed a uterine-specific TSC2-null mouse model where all the mice generate uterine tumors characteristic of LAM and half develop lung metastases. Using RNASeq analysis of uterine tissue from this mouse model, when focusing on genes regulated by estrogen and TSC2, we discovered significant upregulation of inflammatory proteases such as Neutrophil Elastase (NE). NE is secreted by myeloid cells such as polymorphonuclear cells (PMNs) and has been reported to promote invasion, migration, and proliferation in various cancers. We found this to be true in LAM as well, as depleting myeloid cells with an antibody directed against PMNs, or inhibiting NE with the NE inhibitor, sivelestat, markedly decreased TSC2-null uterine tumor growth. NE is released when PMNs undergo Neutrophil Extracellular Trap release, or NETosis. NETosis has been shown to have a pro-tumorigenic role in various cancers and we are investigating the effects of NETosis in LAM. We have also generated a novel uterine-specific TSC2-null mouse in the background of no NE to determine whether uterine tumor burden and lung metastases are reduced in NE-null mice and if these mice have PMNs capable of undergoing NETosis in the absence of NE. Overall, our results suggest that NE release from PMNs is critical for LAM tumor development and may be a novel target for its treatment.
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