Mass Spectrometry Defines Lysophospholipids as Ligands for Chicken MHCY Class I Molecules.

Chicken (Gallus gallus) MHCY class I molecules are highly polymorphic yet substantially different from polymorphic MHC class I molecules that bind peptide Ags. The binding grooves in MHCY class I molecules are hydrophobic and too narrow to accommodate peptides. An earlier structural study suggested that ligands for MHCY class I might be lipids, but the contents of the groove were not clearly identified. In this study, lysophospholipids have been identified by mass spectrometry as bound in two MHCY class I isoforms that differ substantially in sequence. The two isoforms, YF1*7.1 and YF1*RJF34, differ by 35 aa in the α1 and α2 domains that form the MHC class I ligand binding groove. Lyso-phosphatidylethanolamine (lyso-PE) 18:1 was the dominant lipid identified in YF1*7.1 and YF1*RJF34 expressed as recombinant molecules and renatured with ß2-microglobulin in the presence of a total lipid extract from Escherichia coli. Less frequently detected were lyso-PE 17:1, lyso-PE 16:1, and lysophosphatidylglycerols 17:1 and 16:0. These data provide evidence that lysophospholipids are candidate ligands for MHCY class I molecules. Finding that MHCY class I isoforms differing substantially in sequence bind the same array of lysophospholipids indicates that the amino acid polymorphism that distinguishes MHCY class I molecules is not key in defining ligand specificity. The polymorphic positions lie mostly away from the binding groove and might define specificity in interactions of MHCY class I molecules with receptors that are presently unidentified. MHCY class I molecules are distinctive in bound ligand and in display of polymorphic residues.

Mechanical regulation of cell size, fate, and behavior during asymmetric cell division.

Asymmetric cell division (ACD) is an evolutionary conserved mechanism used by prokaryotes and eukaryotes alike to generate cell diversity. ACD can be manifested in biased segregation of macromolecules or differential partitioning of cell organelles. Cells are also constantly subject to extrinsic or intrinsic mechanical forces, influencing cell behavior and fate. During ACD, cell intrinsic forces generated through the spatiotemporal regulation of the actomyosin cytoskeleton can influence sibling cell size. External mechanical stresses are further translated by transcriptional coactivators or mechanically gated ion channels. Here, we will discuss recent literature, exploring how mechanical cues influence various aspects of ACD and stem cell behavior, and how these mechanical cues contribute to cell fate decisions.

Synthesis, Positron Emission Tomography Imaging, and Therapy of Diabody Targeted Drug Lipid Nanoparticles in a Prostate Cancer Murine Model.

The blood clearance of chemotherapeutic drugs such as doxorubicin (Dox) can be extended by incorporation into lipid nanoparticles (LNPs) and further improved by tumor targeting with antibody fragments. We used positron emission tomography (PET) imaging in a murine prostate cancer model to evaluate tumor targeting of LNPs incorporating Dox and antiprostate-specific membrane antigen (PSMA) diabodies. Dox-LNPs were generated by mixing or covalent attachment to water soluble distearoylphosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG)2000. Cu-64 PET imaging was performed with DOTA-conjugated Dox, PEG-LNP, or an anti-PSMA site-specific cysteine-diabody. Since the mixture Dox+PEG-LNP was unstable in serum, further studies utilized Dox covalently bound to LNP ± covalently bound DOTA-cys-diabody (cys-DB)-LNP. Blood clearance of covalent Dox-PEG-LNP was slower than Dox alone or Dox+PEG-LNP. PET imaging of 64Cu-DOTA-Dox-PEG-LNP reached a maximum of 10% ID/g in tumors compared with 3% ID/g of 64Cu-DOTA-Dox, due to the prolonged blood clearance. Mixing 64Cu-DOTA-cys-DB-PEG-LNP with covalent Dox-PEG-LNP gave LNPs containing both drug and tumor targeting cys-DB. The mixed LNPs exhibited increased tumor uptake (15% ID/g) versus untargeted 64Cu-DOTA-Dox-PEG-LNPs (10% ID/g) demonstrating feasibility of the approach. Based on these results, a therapy study with mixed LNPs containing cys-DB-LNP and either Dox-LNP or the antitubulin drug auristatin-LNP showed significant reduction of tumor growth with the auristatin-diabody-LNP mixture, but not the Dox-diabody-LNP mixture.

PET imaging of 64Cu-DOTA-scFv-anti-PSMA lipid nanoparticles (LNPs): Enhanced tumor targeting over anti-PSMA scFv or untargeted LNPs.

INTRODUCTION: Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. METHODS: An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). RESULTS: The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. CONCLUSIONS: The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to an LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery.

Functional mechanisms underlying pleiotropic risk alleles at the 19p13.1 breast-ovarian cancer susceptibility locus.

A locus at 19p13 is associated with breast cancer (BC) and ovarian cancer (OC) risk. Here we analyse 438 SNPs in this region in 46,451 BC and 15,438 OC cases, 15,252 BRCA1 mutation carriers and 73,444 controls and identify 13 candidate causal SNPs associated with serous OC (P=9.2 × 10(-20)), ER-negative BC (P=1.1 × 10(-13)), BRCA1-associated BC (P=7.7 × 10(-16)) and triple negative BC (P-diff=2 × 10(-5)). Genotype-gene expression associations are identified for candidate target genes ANKLE1 (P=2 × 10(-3)) and ABHD8 (P<2 × 10(-3)). Chromosome conformation capture identifies interactions between four candidate SNPs and ABHD8, and luciferase assays indicate six risk alleles increased transactivation of the ADHD8 promoter. Targeted deletion of a region containing risk SNP rs56069439 in a putative enhancer induces ANKLE1 downregulation; and mRNA stability assays indicate functional effects for an ANKLE1 3'-UTR SNP. Altogether, these data suggest that multiple SNPs at 19p13 regulate ABHD8 and perhaps ANKLE1 expression, and indicate common mechanisms underlying breast and ovarian cancer risk.

Molecular Analysis of Mixed Endometrioid and Serous Adenocarcinoma of the Endometrium.

BACKGROUND: The molecular biology and cellular origins of mixed type endometrial carcinomas (MT-ECs) are poorly understood, and a Type II component of 10 percent or less may confer poorer prognoses. METHODOLOGY/PRINCIPAL FINDINGS: We studied 10 cases of MT-EC (containing endometrioid and serous differentiation), 5 pure low-grade endometrioid adenocarcinoma (EAC) and 5 pure uterine serous carcinoma (USC). Endometrioid and serous components of the MT-ECs were macrodissected and the expression of 60 candidate genes compared between MT-EC, pure USC and pure EAC. We found that four genes were differentially expressed when MT-ECs were compared to pure low-grade EAC: CDKN2A (P = 0.006), H19 (P = 0.010), HOMER2 (P = 0.009) and TNNT1 (P = 0.006). Also while we found that even though MT-ECs closely resembled the molecular profiles of pure USCs, they also exhibit lower expression of PAX8 compared to all pure cases combined (P = 0.035). CONCLUSION: Our data suggest that MT-EC exhibits the closest molecular and epidemiological similarities to pure USC and supports clinical observations that suggest patients with MT-EC should receive the same treatment as patients with pure serous carcinoma. Novel specific markers of MT-EC could be of diagnostic utility and could represent novel therapeutic targets in the future.