Macropinocytosis mediates resistance to loss of glutamine transport in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) metabolism and cell growth uniquely rely on glutamine uptake by the transporter ASCT2. Despite previous data reporting cell growth inhibition after ASCT2 knockdown, we here show that ASCT2 CRISPR knockout is tolerated by TNBC cell lines. Despite the loss of a glutamine transporter and low rate of glutamine uptake, intracellular glutamine steady-state levels were increased in ASCT2 knockout compared to control cells. Proteomics analysis revealed upregulation of macropinocytosis, reduction in glutamine efflux and increased glutamine synthesis in ASCT2 knockout cells. Deletion of ASCT2 in the TNBC cell line HCC1806 induced a strong increase in macropinocytosis across five ASCT2 knockout clones, compared to a modest increase in ASCT2 knockdown. In contrast, ASCT2 knockout impaired cell proliferation in the non-macropinocytic HCC1569 breast cancer cells. These data identify macropinocytosis as a critical secondary glutamine acquisition pathway in TNBC and a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine metabolism for their growth, providing a rationale for targeting of more downstream glutamine metabolism components.

Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity.

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition-fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

Inhibition of guanosine monophosphate synthetase (GMPS) blocks glutamine metabolism and prostate cancer growth.

Glutamine is a critical nutrient in cancer; however, its contribution to purine metabolism in prostate cancer has not previously been determined. Guanosine monophosphate synthetase (GMPS) acts in the de novo purine biosynthesis pathway, utilizing a glutamine amide to synthesize the guanine nucleotide. This study demonstrates that GMPS mRNA expression correlates with Gleason score in prostate cancer samples, while high GMPS expression was associated with decreased rates of overall and disease/progression-free survival. Pharmacological inhibition or knockdown of GMPS significantly decreased cell growth in both LNCaP and PC-3 prostate cancer cells. We utilized [15 N-(amide)]glutamine and [U-13 C5 ]glutamine metabolomics to dissect the pathways involved and despite similar growth inhibition by GMPS knockdown, we show unique metabolic effects across each cell line. Using a PC-3 xenograft mouse model, tumor growth was also significantly decreased after GMPS knockdown, highlighting the importance of glutamine metabolism and providing support for GMPS as a therapeutic target in prostate cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The effects of trehalose glycolipid presentation on cytokine production by GM-CSF macrophages.

Trehalose glycolipids (TGLs) are promising vaccine adjuvants, however effects of glycolipid presentation in the in vitro evaluation, and ultimate selection, of lead vaccine adjuvants are often overlooked. To this end, we synthesised a variety of TGLs and determined how the physicochemical presentation of these lipids influenced the cytokine response by bone marrow derived macrophages (BMMs). The TGLs were presented to wild-type and Mincle-/- BMMs as micellar solutions, coated on plates, coated on beads or surfactant solubilised. Medium to long-chain TGLs, either coated on plates or surfactant solubilised, resulted in the highest BMM activation. Stimulation of BMMs with TGLs coated on beads led to a decreased cytokine response, as compared to TGLs alone. All the TGL responses were Mincle dependent, however the mode of presentation did not have the same effect for each individual TGL. This was most apparent for the C22 trehalose monoester, which showed reduced activity compared to its diester counterpart when presented on a plate, but similar activity to the diester when presented as micelles or on beads. Taken together, our findings support the use of several in vitro assays for selecting lead vaccine adjuvants, particularly if structural differences between the adjuvants are pronounced. Graphical abstract The mode of glycolipid presentation, such as micellar solutions, coated on plates, coated on beads or surfactant solubilised, influences the immune response to trehalose glycolipids.

The many roles of Notch signaling during vertebrate somitogenesis.

The embryonic vertebrate body axis contains serially repeated elements, somites, which form sequentially by budding from a posterior tissue called the presomitic mesoderm (PSM). Somites are the embryonic precursors of the vertebrae, ribs and other adult structures. Many inherited human diseases are characterized by dysregulated somitogenesis, resulting in skeletal abnormalities that are evident at birth. Several of these conditions, including some cases of autosomal recessive familial spondylocostal dysostosis (SCDO), arise from mutations in the Notch signaling pathway, which has been demonstrated to be a key player in the regulation of somitogenesis. Here, we review the functional roles of the Notch pathway in vertebrate segmentation, focusing on its activities in a clock that times the formation of somites, as well as in the patterning and production of epithelial somites.

Putative binding sites for mir-125 family miRNAs in the mouse Lfng 3'UTR affect transcript expression in the segmentation clock, but mir-125a-5p is dispensable for normal somitogenesis.

BACKGROUND: In vertebrate embryos, a "segmentation clock" times somitogenesis. Clock-linked genes, including Lunatic fringe (Lfng), exhibit cyclic expression in the presomitic mesoderm (PSM), with a period matching the rate of somite formation. The clock period varies widely across species, but the mechanisms that underlie this variability are not clear. The half-lives of clock components are proposed to influence the rate of clock oscillations, and are tightly regulated in the PSM. Interactions between Lfng and mir-125a-5p in the embryonic chicken PSM promote Lfng transcript instability, but the conservation of this mechanism in other vertebrates has not been tested. Here, we examine whether this interaction affects clock activity in a mammalian species. RESULTS: Mutation of mir-125 binding sites in the Lfng 3'UTR leads to persistent, nonoscillatory reporter transcript expression in the caudal-most mouse PSM, although dynamic transcript expression recovers in the central PSM. Despite this, expression of endogenous mir-125a-5p is dispensable for mouse somitogenesis. CONCLUSIONS: These results suggest that mir-125a sites in the Lfng 3' untranslated region influence transcript turnover in both mouse and chicken embryos, and support the existence of position-dependent regulatory mechanisms in the PSM. They further suggest the existence of compensatory mechanisms that can rescue the loss of mir-125a-5p in mice. Developmental Dynamics 246:740-748, 2017. © 2017 Wiley Periodicals, Inc.

The epigenetic silencing of the estrogen receptor (ER) by hypermethylation of the ESR1 promoter is seen predominantly in triple-negative breast cancers in Indian women.

The proportion of estrogen receptor (ER)-negative and triple-negative (TN) breast cancer in Indian women is higher than that reported in the West, and this difference persists even after their migration to the West. The causes for this significant difference are not entirely clear. Hypermethylation of the ER promoter, an epigenetic alteration, is known to be one of the mechanisms by which the expression of ER is suppressed. Two thirds of breast cancer specimens from an Indian center tested, using the highly sensitive, methylation-specific polymerase chain reaction (MSP) technique, were reported positive. We have used a quantitative assay, the MethyLight, to better assess the extent of methylation in the ESR1 promoter region in 98 breast cancer tumor specimens from Indian women. In addition, the amount of ER transcripts was determined by quantitative reverse transcriptase polymerase chain reaction. Using the stringent cutoff of at least 4% of the target sequence being methylated, 27% of TN tumors were methylated. In addition they demonstrated the highest levels of methylation. In contrast less than 2% ER-positive tumors were hypermethylated. While the proportion of hypermethylated tumors are lower in this study than that estimated using MSP, our results support the notion of increased epigenetic deregulations in ER-negative tumors in general and TN tumors in particular. The development of this assay also permits a rational approach to the selection of patients for clinical trials examining the efficacy of demethylating agents in the treatment of ER-negative breast cancer.

Glutamine addiction promotes glucose oxidation in triple-negative breast cancer.

Glutamine is a conditionally essential nutrient for many cancer cells, but it remains unclear how consuming glutamine in excess of growth requirements confers greater fitness to glutamine-addicted cancers. By contrasting two breast cancer subtypes with distinct glutamine dependencies, we show that glutamine-indispensable triple-negative breast cancer (TNBC) cells rely on a non-canonical glutamine-to-glutamate overflow, with glutamine carbon routed once through the TCA cycle. Importantly, this single-pass glutaminolysis increases TCA cycle fluxes and replenishes TCA cycle intermediates in TNBC cells, a process that achieves net oxidation of glucose but not glutamine. The coupling of glucose and glutamine catabolism appears hard-wired via a distinct TNBC gene expression profile biased to strip and then sequester glutamine nitrogen, but hampers the ability of TNBC cells to oxidise glucose when glutamine is limiting. Our results provide a new understanding of how metabolically rigid TNBC cells are sensitive to glutamine deprivation and a way to select vulnerable TNBC subtypes that may be responsive to metabolic-targeted therapies.

Synthesis of bilocularin A carbamate derivatives and their evaluation as leucine transport inhibitors in prostate cancer cells.

Large-scale extraction of the leaves of the Australian rainforest tree Maytenus bilocularis followed by extensive purification studies afforded the targeted and abundant dihydro-ß-agarofuran, bilocularin A, in sufficient quantities (>500 mg) for detailed semi-synthetic chemistry. Eight bilocularin A carbamate analogues were synthesised using a series of commercially available isocyanate reagents in high purity (>95%) and variable yields (9-91%). All previously undescribed analogues were spectroscopically characterised using NMR, UV, IR and MS data. One compound afforded crystalline material and subsequent single crystal X-ray analysis (Cu-Kα) confirmed the chemical structure along with the absolute configuration. All compounds were evaluated for anti-proliferative activity against the human prostate cancer cell line LNCaP; none of the compounds showed significant (>50%) growth inhibition at 20 µM. Compounds were also tested for their ability to inhibit leucine transport in LNCaP cells, and two analogues showed moderate activity with IC50 values of 8.9 and 8.5 µM. This is the first reported synthesis of dihydro-ß-agarofuran carbamate derivatives.

MSU Crystals Enhance TDB-Mediated Inflammatory Macrophage IL-1ß Secretion.

The tumour microenvironment predominantly consists of macrophages with phenotypes ranging from pro-inflammatory (M1-like) to anti-inflammatory (M2-like). Trehalose-6,6'-dibehenate (TDB) displays moderate anti-tumour activity and stimulates M1-like macrophages via the macrophage inducible C-type lectin (Mincle) resulting in IL-1ß production. In this study, we examined if monosodium urate (MSU), a known vaccine adjuvant, can boost IL-1ß production by TDB-stimulated macrophages. We investigated the effect of MSU/TDB co-treatment on IL-1ß production by GM-CSF (M1-like) and M-CSF/IL-4 (M2-like) differentiated mouse bone marrow macrophages (BMMs) and found that MSU/TDB co-treatment of GM-CSF BMMs significantly enhanced IL-1ß production in a Mincle-dependent manner. Western blot analysis showed that increased IL-1ß production by GM-CSF BMMs was associated with the induction of pro-IL-1ß expression by TDB rather than MSU. Flow cytometry analysis showed that MSU/TDB co-stimulation of GM-CSF BMMs led to greater expansion of CD86high/MHC IIhigh and CD86low/MHC IIlow subpopulations; however, only the latter showed increased production of IL-1ß. Together, these findings provide evidence of the potential to use MSU/TDB co-treatment to boost IL-1ß-mediated anti-tumour activity in M1-like tumour-associated macrophages.

Separate quality-control measures are necessary for estimation of RNA and methylated DNA from formalin-fixed, paraffin-embedded specimens by quantitative PCR.

Estimations of RNA abundance and DNA methylation by quantitative PCR (qPCR) from formalin-fixed, paraffin-embedded (FFPE) tissue specimens are not yet routine in clinical laboratory practice. Excluding specimens with poorly preserved nucleic acids is an important quality-control step for avoiding unreliable results. Because the assays for RNA abundance and DNA methylation have different critical limiting factors, we examined the extent of overlap of excluded specimens for RNA abundance versus methylated DNA. The transcript abundance of three reference genes and of the test gene, estrogen receptor 1 (ESR1), was estimated by SYBR Green qPCR in 250 breast cancer specimens. The estrogen receptor (ER) protein was identified by IHC, and concordance between ESR1 and ER was estimated by Cohen's κ. TaqMan PCR for the ALU-C4 sequence was performed with bisulfite-treated DNA to determine usability in the MethyLight assay. Excluding specimens with mean reference gene CT values exceeding the group mean by >1 SD led to significant improvement of the concordance of ESR1 and ER. Specimens with usable DNA after bisulfite treatment likewise had ALU-C4 CT values of less than the group mean + 1 SD. Samples with low-quality RNA and DNA were partly nonoverlapping. RNA and DNA extracted from the same FFPE block need separate exclusion criteria for qPCR assays of transcript abundance and methylated DNA.

Mir-125a-5p-mediated regulation of Lfng is essential for the avian segmentation clock.

Somites are embryonic precursors of the axial skeleton and skeletal muscles and establish the segmental vertebrate body plan. Somitogenesis is controlled in part by a segmentation clock that requires oscillatory expression of genes including Lunatic fringe (Lfng). Oscillatory genes must be tightly regulated at both the transcriptional and posttranscriptional levels for proper clock function. Here, we demonstrate that microRNA-mediated regulation of Lfng is essential for proper segmentation during chick somitogenesis. We find that mir-125a-5p targets evolutionarily conserved sequences in the Lfng 3' UTR and that preventing interactions between mir-125a-5p and Lfng transcripts in vivo causes abnormal segmentation and perturbs clock activity. This provides strong evidence that microRNAs function in the posttranscriptional regulation of oscillatory genes in the segmentation clock. Further, this demonstrates that the relatively subtle effects of microRNAs on target genes can have broad effects in developmental situations that have critical requirements for tight posttranscriptional regulation.