Glycoprotein non-metastatic melanoma protein B promotes tumor growth and is a biomarker for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including glycoprotein non-metastatic melanoma protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB's unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here, we establish that GPNMB expression is dependent on mTORC1 signaling, and that GPNMB regulates TSC2-null tumor cell invasion in vitro. Further, we demonstrate that GPNMB enhances TSC2-null xenograft tumor growth in vivo, and that ectodomain release is required for this xenograft growth. We also show that GPNMB's ectodomain is released from the cell surface of TSC2-null cells by proteases ADAM10 and 17, and we identify the protease target sequence on GPNMB. Finally, we demonstrate that GPNMB's ectodomain is present at higher levels in LAM patient serum compared to healthy controls and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker.

Lymphangioleiomyomatosis: where endocrinology, immunology and tumor biology meet.

Abstract: 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: mechanistic target of rapamycin complex 1 (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 the 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.

Estradiol Augments Tumor-Induced Neutrophil Production to Promote Tumor Cell Actions in Lymphangioleiomyomatosis Models.

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 cell lines demonstrate modest estradiol (E2) responses, suggesting E2 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 E2-sensitive LAM mouse model. We therefore hypothesized that E2 stimulates tumor growth in part by promoting neutrophil production. Here we report that E2-enhanced lung colonization of TSC2-null cells is indeed dependent on neutrophils. We demonstrate that E2 induces granulopoiesis via estrogen receptor α 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 E2-sensitive neutrophil production. Last, 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 E2 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.

Prolactin is Expressed in Uterine Leiomyomas and Promotes Signaling and Fibrosis in Myometrial Cells.

Uterine leiomyomas are benign, estrogen-sensitive, fibrotic smooth muscle cell tumors occurring in the uterine myometrium. Leiomyomas are a considerable health burden, with a lifetime prevalence of 80% and limited treatment options. Estrogen and progesterone have positive effects on leiomyoma growth, but little is known about the roles of other hormones. One hormone of interest is prolactin, as it has been described to be present and functional in leiomyomas. The current study investigates prolactin production within leiomyomas and its effects on myometrial cells. RNA isolation and quantitative-PCR of human leiomyoma samples relative to matched adjacent myometrium confirms significant expression of prolactin and dopamine receptor D2, a known regulator of prolactin production and release in the pituitary, with no difference in prolactin receptor expression. Immunohistochemistry confirms increased prolactin in leiomyomas compared to adjacent myometrium and uteri from women without leiomyomas. These results suggest that leiomyomas contain cells that produce prolactin, which may then promote signaling in leiomyoma cells to regulate leiomyoma development/growth. Accordingly, we find that prolactin robustly activates STAT5 and MAPK signaling in rat and human myometrial cell lines. Furthermore, prolactin stimulates expression of myofibroblast markers in rat myometrial cells. Our findings suggest that local prolactin production in leiomyomas may stimulate trans-differentiation of myometrial cells to myofibroblasts, which in turn contributes to the fibrotic nature of leiomyomas.

AXL cooperates with EGFR to mediate neutrophil elastase-induced migration of prostate cancer cells.

Neutrophil elastase (NE) promotes multiple stages of tumorigenesis. However, little is known regarding the molecular mechanisms underlying its stimulatory role. This study shows that NE triggers dose-dependent ERK signaling and cell migration in a panel of prostate cell lines representing the spectrum of prostate cell malignancy. All cell lines tested internalize NE; however, NE endocytosis is not required for ERK activation. Instead, NE acts extracellularly by stimulating the release of amphiregulin to initiate EGFR-dependent signaling. Inhibiting amphiregulin's biological activity with neutralizing antibodies, as well as gene silencing of amphiregulin or EGFR, attenuates NE-induced migration in normal and benign prostatic cells. Alternatively, in prostate cancer cells, knockdown of receptor tyrosine kinase AXL, but not EGFR, impairs both basal and NE-stimulated migration. When prostate cells progress to malignancy, the switch from EGFR-to AXL-dependence in NE-mediated migration implies the potential combined application of EGFR and AXL targeted therapy in prostate cancer treatment.

Ligand Binding Prolongs Androgen Receptor Protein Half-Life by Reducing its Degradation.

Androgens are important in female reproduction, but the molecular actions of androgens in female reproductive tissues are not fully understood. We investigated the androgen-responsive transcriptome in human and mouse granulosa cells (GCs) and surprisingly found that the gene-regulation activity of androgen receptor (AR) in these cells is negligible. We then investigated extranuclear actions of AR and found that in human and mouse GCs, as well as in prostate cancer cells, dihydrotestosterone (DHT) dramatically increases the half-life of its own receptor protein. Using the human granulosa-like KGN cells, we show that this effect is not the result of increased AR gene transcription or protein synthesis, nor is it fully abrogated by proteasome inhibition. Knockdown of PTEN, which contributes to degradation of cytoplasmic AR, did not diminish AR accumulation in the presence of DHT. Using immunofluorescence cellular localization studies, we show that nuclear AR is selectively protected from degradation in the presence of DHT. Knockdown of importin 7 expression, a potential regulator of AR nuclear import, does not affect DHT-mediated nuclear accumulation of AR, suggesting importin 7-independent nuclear import of AR in GCs. Further, DNA binding is not required for this protective mechanism. In summary, we show that ligand binding sequesters AR in the nucleus through enhanced nuclear localization independent of DNA binding, thereby protecting it from proteasome degradation in the cytoplasm. This phenomenon distinguishes AR from other sex steroid receptors and may have physiological significance through a positive feedback loop in which androgen induces its own activity in male and female reproductive tissues.

Publish or Perish: Five Steps to Navigating a Less Painful Peer Review.

This Perspective presents comments intended for junior researchers by Carol A. Lange, Editor-in-Chief, Endocrinology, and Stephen R. Hammes, former Editor-in-Chief, Molecular Endocrinology, and former co-Editor-in-Chief, Endocrinology. PRINCIPAL POINTS: 1. Know when you are ready and identify your target audience.2. Select an appropriate journal.3. Craft your title and abstract to capture your key words and deliver your message.4. Tell a clear and impactful story.5. Review, polish, and perfect your manuscript.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Neutrophil elastase from myeloid cells promotes TSC2-null tumor growth.

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.

Compensation, Productivity, and Other Demographics of Academic Divisions of Endocrinology, Diabetes, and Metabolism.

The landscape for academic endocrinology divisions has continued to evolve rapidly;thus, finding reliable data that can be used as benchmarks has become more difficult. Resources are available for salary and relative value units, with the Association of American Medical Colleges, Medical Group Management Association, and Faculty Practice Solutions Center the most commonly used databases. However, details regarding how these data are collected and what they include are unclear. For example, does the income include bonus and/or incentive payments? How are work relative value units defined (individual rendering vs supervising advanced practitioners or fellows or residents)? How is a clinical full-time equivalent defined? In addition, other important data that would be relevant to running an academic division of endocrinology are not available from these, or any other resources, including support staff numbers and compensation or fellowship funding and training information. Therefore, an unmet need exists for reliable data that divisions can use to help shape their visions and goals. To address this demand, the Association of Endocrine Chiefs and Directors, in collaboration with the Endocrine Society, developed a detailed survey for members to address the financial, productivity, composition, and educational issues that they regularly face. Twenty academic institutions throughout the United States completed in the survey in 2018. In the present report, we have provided the results of the survey and some initial interpretations of the findings. Our hope is that the information presented will prove useful as academic endocrinology divisions continue to evolve.

Paxillin regulated genomic networks in prostate cancer.

Paxillin is extensively involved in focal adhesion signaling and kinase signaling throughout the plasma membrane and cytoplasm. However, recent studies in prostate cancer suggest that paxillin also plays a critical role in regulating gene expression within the nucleus, serving as a liaison between cytoplasmic and nuclear MAPK and Androgen Receptor (AR) signaling. Here we used RNA-seq to examine the paxillin-regulated transcriptome in several human prostate cancer cell lines. First, we examined paxillin effects on androgen-mediated transcription in control or paxillin-depleted AR-positive LNCaP and C4-2 human prostate cancer cells. In androgen-dependent LNCaP cells, we found over 1000 paxillin-dependent androgen-responsive genes, some of which are involved in endocrine therapy resistance. Most paxillin-dependent AR-mediated genes in LNCaP cells were no longer paxillin-dependent in androgen-sensitive, castration-resistant C4-2 cells, suggesting that castration-resistance may markedly alter paxillin effects on genomic AR signaling. To examine the paxillin-regulated transcriptome in the absence of androgen signaling, we performed RNA-seq in AR-negative PC3 human prostate cancer cells. Paxillin enhanced several pro-proliferative pathways, including the CyclinD/Rb/E2F and DNA replication/repair pathways. Additionally, paxillin suppressed pro-apoptotic genes, including CASP1 and TNFSF10. Quantitative PCR confirmed that these pathways are similarly regulated by paxillin in LNCaP and C4-2 cells. Functional studies showed that, while paxillin stimulated cell proliferation, it had minimum effect on apoptosis. Thus, paxillin appears to be an important transcriptional regulator in prostate cancer, and analysis of its transcriptome might lead to novel approaches toward the diagnosis and treatment of this important disease.

Impact of estrogens in males and androgens in females.

Androgens and estrogens are known to be critical regulators of mammalian physiology and development. While these two classes of steroids share similar structures (in general, estrogens are derived from androgens via the enzyme aromatase), they subserve markedly different functions via their specific receptors. In the past, estrogens such as estradiol were thought to be most important in the regulation of female biology, while androgens such as testosterone and dihydrotestosterone were believed to primarily modulate development and physiology in males. However, the emergence of patients with deficiencies in androgen or estrogen hormone synthesis or actions, as well as the development of animal models that specifically target androgen- or estrogen-mediated signaling pathways, have revealed that estrogens and androgens regulate critical biological and pathological processes in both males and females. In fact, the concept of "male" and "female" hormones is an oversimplification of a complex developmental and biological network of steroid actions that directly impacts many organs. In this Review, we will discuss important roles of estrogens in males and androgens in females.

Physiological and Pathological Androgen Actions in the Ovary.

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.

Epigenetic Suppression of SERPINB1 Promotes Inflammation-Mediated Prostate Cancer Progression.

Granulocytic myeloid infiltration and resultant enhanced neutrophil elastase (NE) activity is associated with poor outcomes in numerous malignancies. We recently showed that NE expression and activity from infiltrating myeloid cells was high in human prostate cancer xenografts and mouse Pten-null prostate tumors. We further demonstrated that NE directly stimulated human prostate cancer cells to proliferate, migrate, and invade, and inhibition of NE in vivo attenuated xenograft growth. Interestingly, reduced expression of SERPINB1, an endogenous NE inhibitor, also correlates with diminished survival in some cancers. Therefore, we sought to characterize the role of SERPINB1 in prostate cancer. We find that SERPINB1 expression is reduced in human metastatic and locally advanced disease and predicts poor outcome. SERPINB1 is also reduced in Pten-null mouse prostate tumors compared with wild-type prostates, and treatment with sivelestat (SERPINB1 pharmacomimetic) attenuates tumor growth. Knockdown of highly expressed SERPINB1 in nonmalignant prostatic epithelial cells (RWPE-1) increases proliferation, decreases apoptosis, and stimulates expression of epithelial-to-mesenchymal transition markers. In contrast, stable SERPINB1 expression in normally low-expressing prostate cancer cells (C4-2) reduces xenograft growth in vivo. Finally, EZH2-mediated histone (H3K27me3) methylation and DNA methyltransferase-mediated DNA methylation suppress SERPINB1 expression in prostate cancer cells. Analysis of The Cancer Genome Atlas and pyrosequencing demonstrate hypermethylation of the SERPINB1 promoter in prostate cancer compared with normal tissue, and the extent of promoter methylation negatively correlates with SERPINB1 mRNA expression. IMPLICATIONS: Our findings suggest that the balance between SERPINB1 and NE is physiologically important within the prostate and may serve as a biomarker and therapeutic target in prostate cancer.

Bilateral Pheochromocytomas in a Patient with Y175C Von Hippel-Lindau Mutation.

Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is characterized by metachronously occurring tumors including pheochromocytoma, renal cell carcinoma (RCC), and hemangioblastoma. Although VHL disease leads to reduced life expectancy, its diagnosis is often missed and tumor screening guidelines are sparse. VHL protein acts as a tumor suppressor by targeting hypoxia-inducible factors (HIFs) for degradation through an oxygen-dependent mechanism. VHL mutants with more severely reduced HIF degrading function carry a high risk of RCC, while mutants with preserved HIF degrading capacity do not cause RCC but still lead to other tumors. VHL disease is classified into clinical types (1 and 2A-2C) based on this genotype-phenotype relationship. We report a case of bilateral pheochromocytomas and no other VHL-related tumors in a patient with Y175C VHL and show that this mutant preserves the ability to degrade HIF in normal oxygen conditions but, similar to the wild-type VHL protein, loses its ability to degrade HIF under hypoxic conditions. This study adds to the current understanding of the structure-function relationship of VHL mutations, which is important for risk stratification of future tumor development in the patients.

Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB) and Cancer: A Novel Potential Therapeutic Target.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a transmembrane protein enriched on the cell surface of cancer cells, including melanoma, glioblastoma, and triple-negative breast cancer. There is growing evidence identifying GPNMB as a tumor-promoter; however, despite its biological and clinical significance, the molecular mechanisms engaged by GPNMB to promote tumorigenesis are not well understood. GPNMB promotes aggressive behaviors such as tumor cell proliferation, migration, and invasion. The extracellular domain of GPNMB shed from the cell surface interacts with integrins to facilitate in the recruitment of immune-suppressive and pro-angiogenic cells to the tumor microenvironment, thereby enhancing tumor migration and invasion. GPNMB also modulates receptor tyrosine kinases and integrin signaling in a cell autonomous fashion, leading to downstream kinase signaling that in turn triggers the expression and secretion of tumorigenic factors such as matrix metalloproteinases (MMPs) and cytokines. Therefore, GPNMB exerts its pro-tumorigenic role both intracellularly and in a paracrine fashion through shedding its extracellular domain. This review highlights the importance of GPNMB in cancer progression and discusses molecular mediators of GPNMB-induced tumor growth and invasion.