We have demonstrated the selective secretion of high mol wt PRL series (big big PRL) in women with hyperprolactinemia and normal ovarian function. This observation suggests that big big PRL is immunologically similar, but biologically less active, than monomeric or little PRL. In this study we determined the molecular size heterogeneity of immunoreactive PRL in the serum from two ovulatory hyperprolactinemic women (subjects A and B) who had large amounts of serum big big PRL during a menstrual cycle and/or gestation. Serum samples obtained throughout the menstrual cycle (days 6, 10, 14, 17, 23, and 28, taking as day 1 the first day of bleeding) and pregnancy (weeks 7, 9, 11, 15, 20, 25, 30, 34, and 38) were fractionated by gel filtration chromatography. PRL was identified in column eluates by specific RIA. Two additional pregnant women, one with a bromocriptine-treated PRL-secreting adenoma (subject C), and a normal woman (subject D) were studied. Big big PRL was the predominant species throughout the different phases of the menstrual cycle in subject B, comprising 70-80% of the total immunoreactive PRL. Most of the remainder was big PRL, and little PRL was present in only small amounts (6-12%) during the luteal phase. During their pregnancies, the serum PRL in subjects A and B initially was mostly big big PRL, but later in gestation the PRL composition shifted from the high mol wt variants to little PRL. The infant's cord (subject A) and peripheral (subject B) serum at birth contained appreciable quantities of big big and big PRL, respectively. These results indicate that structural changes in PRL occur during pregnancy and the menstrual cycle which are probably influenced by the hormonal environment. In addition, the occurrence of larger mol wt PRL species in the serum of the infant of a hyperprolactinemic mother suggests that the presence of high proportions of big big PRL in the serum is genetically determined.
Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies.
Skeletal muscle morphogenesis depends upon interactions between developing muscle fibers and the extracellular matrix (ECM) that anchors fibers to the myotendinous junction (MTJ). The pathways that organize the ECM and regulate its engagement by cell-matrix adhesion complexes (CMACs) are therefore essential for muscle integrity. Here, we demonstrate the impact of transmembrane protein 2 (tmem2) on cell-matrix interactions during muscle morphogenesis in zebrafish. Maternal-zygotic tmem2 mutants (MZtmem2) exhibit muscle fiber detachment, in association with impaired laminin organization and ineffective fibronectin degradation at the MTJ. Similarly, disorganized laminin and fibronectin surround MZtmem2 cardiomyocytes, which could account for their hindered movement during cardiac morphogenesis. In addition to ECM defects, MZtmem2 mutants display hypoglycosylation of α-dystroglycan within the CMAC, which could contribute to the observed fiber detachment. Expression of the Tmem2 ectodomain can rescue aspects of the MZtmem2 phenotype, consistent with a possible extracellular function of Tmem2. Together, our results suggest that Tmem2 regulates cell-matrix interactions by affecting both ECM organization and CMAC activity. These findings evoke possible connections between the functions of Tmem2 and the etiologies of congenital muscular dystrophies, particularly dystroglycanopathies.
Background Atrioventricular valve development relies upon the precisely defined dimensions of the atrioventricular canal (AVC). Current models suggest that Wnt signaling plays an important role atop a pathway that promotes AVC development. The factors that confine AVC differentiation to the appropriate location, however, are less well understood. Results Transmembrane protein 2 (Tmem2) is a key player in restricting AVC differentiation: in zebrafish, tmem2 mutants display an expansion of AVC characteristics, but the molecular mechanism of Tmem2 function in this context remains unclear. Through structure‐function analysis, we demonstrate that the extracellular portion of Tmem2 is crucial for its role in restricting AVC boundaries. Importantly, the Tmem2 ectodomain contains regions implicated in the depolymerization of hyaluronic acid (HA). We find that tmem2 mutant hearts exhibit excess HA deposition alongside broadened distribution of Wnt signaling. Moreover, addition of ectopic hyaluronidase can restore the restriction of AVC differentiation in tmem2 mutants. Finally, we show that alteration of a residue important for HA depolymerization impairs the efficacy of Tmem2 function during AVC development. Conclusions Taken together, our data support a model in which HA degradation, regulated by Tmem2, limits the distribution of Wnt signaling and thereby confines the differentiation of the AVC.
Spatially resolved gene expression has emerged as a crucial technique to understand complex multicellular interactions within the tumor and its microenvironment. Interrogation of complex cellular interactions within the tumor microenvironment (TME) requires a multi-omics approach where multiple RNA and protein targets can be visualized within the same tumor sample and be feasible in FFPE sample types. Simultaneous detection of RNA and protein can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. Examination of RNA by in situ hybridization (ISH) and protein by immunohistochemistry (IHC) or immunofluorescence (IF) are widely used and accepted techniques for the detection of biomarkers in tumor samples. Given the similarities in workflow, co-detection of RNA and protein by combining ISH and IHC/IF in a single assay can be a powerful multi-omics solution for interrogating the complex tumor and its microenvironment. In this report we combined the single cell, single molecule RNA ISH technology known as RNAscope with IHC/IF to simultaneously detect RNA and protein in the same FFPE tumor section using both chromogenic and fluorescence detection methods. We demonstrate co-localization of target mRNA and the corresponding protein in human cancer samples, visualize infiltration of immune cells into the TME, characterize the activation state of immune cells in the TME, identify single cell gene expression within cellular boundaries demarcated by IHC/IF, examine cell type-specific expression of multiple immune checkpoint markers, and distinguish endogenous T cells from activated CAR+ T cells. Overall, we show that co-detection of RNA by the RNAscope ISH assay and protein by the IHC/IF assay in the same FFPE section is a feasible methodology. The combined RNAscope ISH-IHC/IF workflow is a powerful technique that can be used to study gene expression signatures at the RNA and protein level with spatial and single cell resolution. By leveraging the strength of the similar workflows of RNAscope ISH and IHC/IF assays, this methodology combines transcriptomics and proteomics in the same tissue section, providing a multi-omics approach for characterizing complex tissues and revealing cell type specific gene expression with spatial and single cell resolution. Citation Format: Anushka Dikshit, Jyoti Phatak, Siobhan Kernag, Helly Pimental, Hailing Zong, Courtney Todorov, Lydia Hernandez, Jeffrey Kim, Bingqing Zhang, Courtney Anderson, Xiao-Jun Ma. Spatially resolve RNA and protein simultaneously in FFPE tumor samples by combining RNAscope in situ hybridization and immunohistochemistry assays [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2706.
Head and neck squamous cell carcinoma (HNSCC) is the most prevalent cancer affecting the upper aerodigestive tract, with an annual incidence of 600,000 patients and a five year survival of approximately 60% worldwide. Molecular mechanisms driving the development of HNSCC have recently begun to be discovered, with The Cancer Genome Atlas (TCGA) uncovering the genomic landscape of 279 cases of HNSCC. Alterations in cell death pathways were commonly found in the TCGA analysis, with ∼30% of samples harboring 11q13/22 amplifications and overexpression of genes encoding for Fas-associated death domain (FADD) and/or cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2). While overexpression of cIAP1 has been implicated in resistance to cytotoxic therapies, the role of FADD amplification as a target for therapy and in mechanisms of cell death is not well understood. Birinapant is a novel second mitochondria-derived activator of caspases (SMAC)-mimetic that targets and promotes degradation of cIAPs. Its clinical efficacy is currently being investigated in phase II trials of patients with ovarian cancer and leukemia. However, its preclinical and clinical efficacies have not been tested in HNSCC and genomic markers of sensitivity remain to be defined. Here we hypothesized that overexpression of FADD and cIAP1/2 could modulate birinapant sensitivity in HNSCC. To test this hypothesis, we have treated a panel of 11 HPV(-) and 8 HPV(+) HNSCC cell lines with birinapant alone and in combination with death agonists TNFα or TRAIL. UMSCC-46, an HPV(-) cell line which possesses high FADD expression, was the only cell line to reach half maximal inhibitory concentration (IC50) 72 hours post treatment with birinapant alone (IC50 = 10.7 nM); however, 8 of 11 HPV(-) cell lines and all 8 HPV(+) cell lines attained an IC50 (range: 0.1 - 794 nM) when treated with birinapant in combination with either TNFα or TRAIL. We further demonstrated that forced FADD overexpression in a previously resistant cell line (UMSCC-38) led to sensitization when treated with birinapant and TNFα. In vivo, two FADD/cIAP1 overexpressing murine xenograft models of HNSCC, UMSCC-46 and UMSCC-11B, were treated with birinapant at 15 mg/kg or 30 mg/kg every 3 days for a total of 10 treatments. The single modality regimen led to tumor growth inhibition and prolonged host survival. Additionally, combination treatment with birinapant 15 mg/kg and radiation 2Gy/day M-F for 2 weeks synergistically induced TNFα and led to a cure of animals bearing UMSCC-46 xenografts. Mechanistically, birinapant enhanced degradation of cIAP1 and modulated caspase apoptotic or MLKL necroptotic cell death markers in vitro and in vivo. These results suggest that patients harboring genomic alterations in FADD and/or cIAP overexpression may be candidates for treatment with birinapant and radiation. Supported by NIDCD intramural projects ZIA-DC-000073, and 74. Citation Format: Adeeb Derakhshan, Danielle Eytan, Grace Snow, Sophie Carlson, Anthony Saleh, Hui Cheng, Stephen Schiltz, Suresh Mohan, Shaleeka Cornelius, Jamie Coupar, Anastasia Sowers, Lydia Hernandez, James Mitchell, Christina Annunziata, Zhong Chen, Carter Van Waes. Targeted therapy for head and neck squamous cell carcinoma using the novel SMAC-mimetic birinapant. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3821.
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