The Ephrin B2 Receptor Tyrosine Kinase Is a Regulator of Proto-oncogene MYC and Molecular Programs Central to Barrett's Neoplasia.

BACKGROUND & AIMS: Mechanisms contributing to the onset and progression of Barrett's (BE)-associated esophageal adenocarcinoma (EAC) remain elusive. Here, we interrogated the major signaling pathways deregulated early in the development of Barrett's neoplasia. METHODS: Whole-transcriptome RNA sequencing analysis was performed in primary BE, EAC, normal esophageal squamous, and gastric biopsy tissues (n = 89). Select pathway components were confirmed by quantitative polymerase chain reaction in an independent cohort of premalignant and malignant biopsy tissues (n = 885). Functional impact of selected pathway was interrogated using transcriptomic, proteomic, and pharmacogenetic analyses in mammalian esophageal organotypic and patient-derived BE/EAC cell line models, in vitro and/or in vivo. RESULTS: The vast majority of primary BE/EAC tissues and cell line models showed hyperactivation of EphB2 signaling. Transcriptomic/proteomic analyses identified EphB2 as an endogenous binding partner of MYC binding protein 2, and an upstream regulator of c-MYC. Knockdown of EphB2 significantly impeded the viability/proliferation of EAC and BE cells in vitro/in vivo. Activation of EphB2 in normal esophageal squamous 3-dimensional organotypes disrupted epithelial maturation and promoted columnar differentiation programs, notably including MYC. EphB2 and MYC showed selective induction in esophageal submucosal glands with acinar ductal metaplasia, and in a porcine model of BE-like esophageal submucosal gland spheroids. Clinically approved inhibitors of MEK, a protein kinase that regulates MYC, effectively suppressed EAC tumor growth in vivo. CONCLUSIONS: The EphB2 signaling is frequently hyperactivated across the BE-EAC continuum. EphB2 is an upstream regulator of MYC, and activation of EphB2-MYC axis likely precedes BE development. Targeting EphB2/MYC could be a promising therapeutic strategy for this often refractory and aggressive cancer.

Temporal Dynamics of Subclinical Malaria in Different Transmission Zones of Myanmar.

Countries in the Greater Mekong Subregion have committed to eliminate Plasmodium falciparum malaria by 2025. Subclinical malaria infections that can be detected by highly sensitive polymerase chain reaction (PCR) testing in asymptomatic individuals represent a potential impediment to this goal, although the extent to which these low-density infections contribute to transmission is unclear. To understand the temporal dynamics of subclinical malaria in this setting, a cohort of 2,705 participants from three epidemiologically distinct regions of Myanmar was screened for subclinical P. falciparum and P. vivax infection using ultrasensitive PCR (usPCR). Standard rapid diagnostic tests (RDTs) for P. falciparum were also performed. Individuals who tested positive for malaria by usPCR were followed for up to 12 weeks. Regression analysis was performed to estimate whether the baseline prevalence of infection and the count of repeated positive tests were associated with demographic, behavioral, and clinical factors. At enrollment, the prevalence of subclinical malaria infection measured by usPCR was 7.7% (1.5% P. falciparum monoinfection, 0.3% mixed P. falciparum and P. vivax, and 6.0% P. vivax monoinfection), while P. falciparum prevalence measured by RDT was just 0.2%. Prevalence varied by geography and was higher among older people and in those with outdoor exposure and travel. No difference was observed in either the prevalence or count of subclinical infection by time of year, indicating that even in low-endemicity areas, a reservoir of subclinical infection persists year-round. If low-density infections are shown to represent a significant source of transmission, identification of high-risk groups and locations may aid elimination efforts.

Parathyroid-Targeted Overexpression of Regulator of G-Protein Signaling 5 (RGS5) Causes Hyperparathyroidism in Transgenic Mice.

The relationship between impaired calcium sensing, dysregulated parathyroid hormone (PTH) secretion, and parathyroid cell proliferation in parathyroid neoplasia is not understood. We previously reported that a GTPase activating protein, regulator of G-protein signaling 5 (RGS5) is overexpressed in a subset of parathyroid tumors associated with primary hyperparathyroidism (PHPT) and that RGS5 can inhibit signaling from the calcium-sensing receptor (CASR). In vivo, we found that RGS5-null mice have abnormally low PTH levels. To gain a better understanding of the potential role of RGS5 overexpression in parathyroid neoplasia and PHPT and to investigate whether inhibition of CASR signaling can lead to parathyroid neoplasia, we created and characterized a transgenic mouse strain overexpressing RGS5 specifically in the parathyroid gland. These mice develop hyperparathyroidism, bone changes reflective of elevated PTH, and parathyroid neoplasia. Further, expression of exogenous RGS5 in normal human parathyroid cells results in impaired signaling from CASR and negative feedback on PTH secretion. These results provide evidence that RGS5 can modulate signaling from CASR and support a role for RGS5 in the pathogenesis of PHPT through inhibition of CASR signaling. © 2019 American Society for Bone and Mineral Research.

Transcriptional profiling reveals distinct classes of parathyroid tumors in PHPT.

The clinical presentation of primary hyperparathyroidism (PHPT) varies widely, although the underlying mechanistic reasons for this disparity remain unknown. We recently reported that parathyroid tumors can be functionally segregated into two distinct groups on the basis of their relative responsiveness to ambient calcium, and that patients in these groups differ significantly in their likelihood of manifesting bone disability. To examine the molecular basis for this phenotypic variation in PHPT, we compared the global gene expression profiles of calcium-sensitive and calcium-resistant parathyroid tumors. RNAseq and proteomic analysis identified a candidate set of differentially expressed genes highly correlated with calcium-sensing capacity. Subsequent quantitative assessment of the expression levels of these genes in an independent cohort of parathyroid tumors confirmed that calcium-sensitive tumors cluster in a discrete transcriptional profile group. These data indicate that PHPT is not an etiologically monolithic disorder and suggest that divergent molecular mechanisms could drive the observed phenotypic differences in PHPT disease course, provenance, and outcome.

Live-Cell Visualization of Calcium Flux in Vibratome-Cut Thick Sections of Viable Tumor Tissue.

This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein-coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include: (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases: (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live-cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.

Impaired calcium sensing distinguishes primary hyperparathyroidism (PHPT) patients with low bone mineral density.

CONTEXT: A subset of PHPT patients exhibit a more severe disease phenotype characterized by bone loss, fractures, recurrent nephrolithiasis, and other dysfunctions, but the underlying reasons for this disparity in clinical presentation remain unknown. OBJECTIVE: We sought to identify new mechanistic indices that could inform more personalized management of PHPT. DESIGN: Pre-, peri-, and postoperative data and demographic, clinical, and pathological information from patients undergoing parathyroidectomy for PHPT were collected. Univariate and partial Spearman correlation was used to estimate the association of parathyroid tumor calcium sensing capacity with select variables. PATIENTS OR OTHER PARTICIPANTS: An unselected series of 237 patients aged >18years and undergoing parathyroidectomy for PHPT were enrolled. MAIN OUTCOME MEASURES: Calcium sensing capacity, expressed as the concentration required for half-maximal biochemical response (EC50), was evaluated in parathyroid tumors from an unselected series of 74 patients and assessed for association with clinical parameters. The hypothesis was that greater disease severity would be associated with attenuated calcium sensitivity and biochemically autonomous parathyroid tumor behavior. RESULTS: Parathyroid tumors segregated into two distinct groups of calcium responsiveness (EC50<3.0 and ≥3.0mM). The low EC50 group (n=27) demonstrated a mean calcium EC50 value of 2.49mM [95% confidence interval (CI): 2.43-2.54mM], consistent with reference normal activity. In contrast, the high EC50 group (n=47) displayed attenuated calcium sensitivity with a mean EC50 value of 3.48mM [95% CI: 3.41-3.55mM]. Retrospective analysis of the clinical registry data suggested that high calcium EC50 patients presented with a more significant preoperative bone mineral density (BMD) deficit with a t-score of -2.7, (95% CI: -3.4 to -1.9) versus 0.9, (95% CI: -2.1 to -0.4) in low EC50 patients (p<0.001). After adjusting for gender, age, BMI, 25 OH vitamin D level and preoperative iPTH, lowest t-score and calcium EC50 were inversely correlated, with a partial Spearman correlation coefficient of -0.35 (p=0.02). CONCLUSIONS: Impaired calcium sensing in parathyroid tumors is selectively observed in a subset of patients with more severe bone mineral density deficit. Assessment of parathyroid tumor biochemical behavior may be a useful predictor of disease severity as measured by bone mineral density in patients with PHPT.

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium-Sensing Receptor-Mediated Signaling.

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and ß-catenin), the molecular basis of abnormal calcium sensing by calcium-sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein-coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3',5'-cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N-terminally truncated human GPR64 is constitutively active and a 15-amino acid-long peptide C-terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full-length GPR64 physically interacts with CaSR and attenuates the CaSR-mediated intracellular Ca2+ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.

Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia.

Parathyroid adenomas (PAs) causing primary hyperparathyroidism (PHPT) are histologically heterogeneous yet have been historically viewed as largely monotypic entities arising from clonal expansion of a single transformed progenitor. Using flow cytometric analysis of resected adenomatous parathyroid glands, we have isolated and characterized chief cells, oxyphil cells, and tumor-infiltrating lymphocytes. The parathyroid chief and oxyphil cells produce parathyroid hormone (PTH), express the calcium-sensing receptor (CASR), and mobilize intracellular calcium in response to CASR activation. Parathyroid tumor infiltrating lymphocytes are T cells by immunophenotyping. Under normocalcemic conditions, oxyphil cells produce ∼50% more PTH than do chief cells, yet display significantly greater PTH suppression and calcium flux response to elevated calcium. In contrast, CASR expression and localization are equivalent in the respective parathyroid cell populations. Analysis of tumor clonality using X-linked inactivation assays in a patient-matched series of intact tumors, preparatively isolated oxyphil and chief cells, and laser-captured microdissected PA specimens demonstrate polyclonality in 5 of 14 cases. These data demonstrate the presence of functionally distinct oxyphil and chief cells within parathyroid primary adenomas and provide evidence that primary PA can arise by both clonal and polyclonal mechanisms. The clonal differences, biochemical activity, and relative abundance of these parathyroid adenoma subpopulations likely reflect distinct mechanisms of disease in PHPT.