Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. ...However, their biological function is unknown. Here, we show that the fly family member Lilipod (Lili) is required for germline stem cell (GSC) self-renewal in theDrosophilaovary where it enhances bone morphogenetic protein (BMP) signaling.lilimutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factorbam.Conversely, overexpressed Lili induces supernumerary pMad-positivebamP-GFP–negative GSCs. Interestingly, differentiation oflilimutant GSCs isbam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing abam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first, to our knowledge, in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.
Breast carcinoma with tubulopapillary features is a newly described entity associated with poor prognosis with only 14 tumors reported in the literature. We report 2 additional tumors and identify ...novel immunohistochemical and molecular features of the tumor. The first tumor was from a 72-year-old woman with nonmetastatic breast carcinoma and the second was from a 32-year-old woman with metastatic breast carcinoma who received neoadjuvant therapy. Both tumors had high-grade nuclear features with a distinctive morphology characterized by infiltrating open glands with intratubular papillary and micropapillary projections in >90% of the invasive carcinoma. In addition to the usual predictors of aggressive behavior, both tumors showed a high expression of p16 and SOX10, which has not been previously described. Targeted tumor sequencing revealed pathogenic variants of TP53 in both tumors, in agreement with previous reports. Prior studies have shown a correlation between p16 and SOX10 expression with high-grade features and worse prognosis; typically seen in triple-negative carcinomas as demonstrated in both of our tumors. However, not all reported tumors of breast carcinoma with tubulopapillary features have demonstrated a triple-negative profile as there are a few reports of tumors with estrogen receptor and/or human epidermal growth factor 2 expression. Due to their distinct morphologic and molecular characteristics, breast carcinoma with tubulopapillary features may represent a new breast cancer histologic subtype.
Invasive micropapillary breast carcinoma (IMPC) is a rare breast cancer subtype characterized by small tumor cell clusters with loss of stromal attachment, an inside-out growth appearance, and ...lymphotropism. IMPC is associated with high incidence of lymphovascular invasion (LVI) and lymph node metastasis. Activated Wnt signaling has been implicated in the metastasis of other aggressive breast tumors, including triple-negative and basal-like carcinomas. In this study, we tested whether activated Wnt signaling could be detected in IMPC. Upon ligand binding, the central mediator of the Wnt pathway, β-catenin, accumulates in the cytosol and translocates to the nucleus where it forms a complex with lymphoid enhancer-binding factor 1 (LEF1) to regulate transcription. We performed immunostaining for β-catenin and LEF1 on a well-annotated cohort of 40 breast tumors and nodal metastases displaying micropapillary histopathology. Strong nuclear accumulation of β-catenin was not observed, however a dim cytosolic and/or nuclear accumulation of β-catenin was sometimes seen in IMPC and this expression pattern was significantly associated with nodal metastasis. β-catenin expression correlated with the upregulation of LEF1 in IMPC. LEF1 expression was detected in 26 of 40 (65%) cases and was specifically enriched at the invasive front of the tumor and in tumor clusters undergoing LVI. Detection of LEF1 expression in the primary tumor was associated with an increased rate of LVI, lymph node metastasis, and disease relapse. LEF1 and β-catenin expression levels were significantly higher in metastases compared with primary tumors. In summary, this study demonstrates an association between the upregulation of β-catenin/LEF1 and the metastasis of IMPC.
BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. ...These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments.
Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system.
Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Leptomeningeal disease (LMD) is a rare form of central nervous system (CNS) metastasis wherein tumor cells invade the cerebrospinal fluid (CSF) filled space that surrounds the brain and ...spinal cord. For patients with LMD, prognosis is extremely poor even with aggressive treatment. The mechanisms of progression to LMD and the adaptations tumor cells make to survive in this metabolically sparse microenvironment are poorly understood. As 60% of patients with LMD have concurrent or prior parenchymal metastases, our laboratory examined our established murine models of parenchymal metastases for signs of leptomeningeal infiltration. We identify two xenograft non-small cell lung cancer (NSCLC) models of intraparenchymal metastasis following intra-arterial injection that show progression to LMD in a subset of cases. In the H2030-BrM3 model, this occurs de novo, whereas the EGFR-mutant PC9-R2 model progresses to LMD only following onset of resistance to tyrosine kinase inhibitor treatment. Subsequent in vivo passaging of the H2030-BrM3 line through the cerebral lateral ventricles resulted in the H2030-LMD2 cell line, which has high affinity for leptomeningeal metastases and shows extensive perivascular invasion within the brain parenchyma. In vitro, the H2030-LMD2 line shows altered clustering behavior and increased survival when cultured in suspension. RNA-sequencing of this cell line across adherent and suspension culture conditions shows multiple biological processes upregulated in the LMD-tropic line, including chemotaxis, apical junction formation, and TGF-β signaling. Targetable pathways emerging from this analysis will be functionally investigated for their role in promoting progression to LMD, including through validation in the syngeneic KPN1-BrM line which has been selected for its CNS affinity and shows progression to LMD. Spatial sequencing and multiplexed immunofluorescence will furthermore investigate the tumor microenvironment in the perivascular niche and subarachnoid spaces. These findings will be clinically corroborated with molecular characterization of the CSF of patients with leptomeningeal metastases.
Efficient DNA replication involves coordinated interactions among DNA polymerase, multiple factors, and the DNA. From bacteriophage T4 to eukaryotes, these factors include a helicase to unwind the ...DNA ahead of the replication fork, a single-stranded binding protein (SSB) to bind to the ssDNA on the lagging strand, and a helicase loader that associates with the fork, helicase, and SSB. The previously reported structure of the helicase loader in the T4 system, gene product (gp)59, has revealed an N-terminal domain, which shares structural homology with the high mobility group (HMG) proteins from eukaryotic organisms. Modeling of this structure with fork DNA has suggested that the HMG-like domain could bind to the duplex DNA ahead of the fork, whereas the C-terminal portion of gp59 would provide the docking sites for helicase (T4 gp41), SSB (T4 gp32), and the ssDNA fork arms. To test this model, we have used random and targeted mutagenesis to generate mutations throughout gp59. We have assayed the ability of the mutant proteins to bind to fork, primed fork, and ssDNAs, to interact with SSB, to stimulate helicase activity, and to function in leading and lagging strand DNA synthesis. Our results provide strong biochemical support for the role of the N-terminal gp59 HMG motif in fork binding and the interaction of the C-terminal portion of gp59 with helicase and SSB. Our results also suggest that processive replication may involve the switching of gp59 between its interactions with helicase and SSB.
Replication requires coordinated interactions among proteins/DNA.
Mutational analysis of T4 helicase loader, gp59, identifies domains that interact with DNA and replication factors.
The high mobility group motif of gp59 interacts with the DNA fork; gp59 C-terminal surfaces interact with helicase and single-stranded binding protein.
Processive replication may involve switching of gp59 between interactions with helicase and the single-stranded binding protein.
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