The phospholipid flippase ATP9A enhances macropinocytosis to promote nutrient starvation tolerance in hepatocellular carcinoma.

Macropinocytosis is an effective strategy to mitigate nutrient starvation. It can fuel cancer cell growth in nutrient-limited conditions. However, whether and how macropinocytosis contributes to the rapid proliferation of hepatocellular carcinoma cells, which frequently experience an inadequate nutrient supply, remains unclear. Here, we demonstrated that nutrient starvation strongly induced macropinocytosis in some hepatocellular carcinoma cells. It allowed the cells to acquire extracellular nutrients and supported their energy supply to maintain rapid proliferation. Furthermore, we found that the phospholipid flippase ATP9A was critical for regulating macropinocytosis in hepatocellular carcinoma cells and that high ATP9A levels predicted a poor outcome for patients with hepatocellular carcinoma. ATP9A interacted with ATP6V1A and facilitated its transport to the plasma membrane, which promoted plasma membrane cholesterol accumulation and drove RAC1-dependent macropinocytosis. Macropinocytosis inhibitors significantly suppressed the energy supply and proliferation of hepatocellular carcinoma cells characterised by high ATP9A expression under nutrient-limited conditions. These results have revealed a novel mechanism that overcomes nutrient starvation in hepatocellular carcinoma cells and have identified the key regulator of macropinocytosis in hepatocellular carcinoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Quasiclassical Trajectory Study of the O(3P) + CO2(1Σg+) Reaction at Hyperthermal Energies.

This paper presents the reaction mechanism, cross sections, and product energy partitioning for the O + CO2 reaction, calculated using Born-Oppenheimer molecular dynamics simulations with the quasiclassical trajectory (BOMD-QCT) method. At collision energies up to 9.5 eV, three reactions, oxygen exchange (above ∼1.5 eV), abstraction (above ∼5.5 eV), and dissociation (above ∼7.5 eV) occur, with abstraction and dissociation involving either an insertion-elimination mechanism or a stripping mechanism. The insertion-elimination mechanism involves the formation of a planar CO3 intermediate which lies 0.52 eV above the ground-state CO2; the energetic barrier for oxygen abstraction via this mechanism is 3.52 eV. Interestingly, the insertion-elimination mechanism predominately contributes to the cross sections at collision energies just above the effective energetic threshold for the abstraction and dissociation reactions; at higher collision energies, the contribution from the stripping mechanism increases and eventually dominates. At a collision energy of 9.5 eV, the cross sections for oxygen exchange, abstraction, and dissociation are 4.17 a02, 1.58 a02, and 0.68 a02, respectively. The lower reaction cross sections, higher effective reaction barrier, and product energy distribution of the stripping mechanism were attributed to the short lifetime (28 fs) of the OCOO species compared with that of the CO3 species (45 fs) that arises in the insertion-elimination mechanism. For the exchange reaction, it is found that roughly 40% of the reactant translational energy ends up in CO2 vibration, which provides a single-collision mechanism to produce highly excited CO2. We also studied intersystem crossing effects using trajectory surface hopping calculations and find no changes compared to single surface (triplet) calculations at energies below 7.5 eV; however, at 7.5 eV and higher the abstraction cross sections are changed by as much as 20%, and the (very small) dissociation cross sections are changed by factors of four or more.

In Situ Nanoscale Redox Mapping Using Tip-Enhanced Raman Spectroscopy.

Electrochemical atomic force microscopy tip-enhanced Raman spectroscopy (EC-AFM-TERS) was used for the first time to spatially resolve local heterogeneity in redox behavior on an electrode surface in situ and at the nanoscale. A structurally well-defined Au(111) nanoplate located on a polycrystalline ITO substrate was studied to examine nanoscale redox contrast across the two electrode materials. By monitoring the TERS intensity of adsorbed Nile Blue (NB) molecules on the electrode surface, TERS maps were acquired with different applied potentials. The EC-TERS maps showed a spatial contrast in TERS intensity between Au and ITO. TERS line scans near the edge of a 20 nm-thick Au nanoplate demonstrated a spatial resolution of 81 nm under an applied potential of -0.1 V vs Ag/AgCl. The intensities from the TERS maps at various applied potentials followed Nernstian behavior, and a formal potential ( E0') map was constructed by fitting the TERS intensity at each pixel to the Nernst equation. Clear nanoscale spatial contrast between the Au and ITO regions was observed in the E0' map. In addition, statistical analysis of the E0' map identified a statistically significant 4 mV difference in E0' on Au vs ITO. Electrochemical heterogeneity was also evident in the E0' distribution, as a bimodal distribution was observed in E0' on polycrystalline ITO, but not on gold. A direct comparison between an AFM friction image and the E0' map resolved the electrochemical behavior of individual ITO grains with a spatial resolution of ∼40 nm. The variation in E0' was attributed to different local surface charges on the ITO grains. Such site-specific electrochemical information with nanoscale spatial and few mV voltage resolutions is not available using ensemble spectroelectrochemical methods. We expect that in situ redox mapping at the nanoscale using EC-AFM-TERS will have a crucial impact on understanding the role of nanoscale surface features in applications such as electrocatalysis.

Tip-Enhanced Raman Excitation Spectroscopy (TERES): Direct Spectral Characterization of the Gap-Mode Plasmon.

The plasmonic properties of tip-substrate composite systems are of vital importance to near-field optical spectroscopy, in particular tip-enhanced Raman spectroscopy (TERS), which enables operando studies of nanoscale chemistry at a single molecule level. The nanocavities formed in the tip-substrate junction also offer a highly tunable platform for studying field-matter interactions at the nanoscale. While the coupled nanoparticle dimer model offers a correct qualitative description of gap-mode plasmon effects, it ignores the full spectrum of multipolar tip plasmon modes and their interaction with surface plasmon polariton (SPP) excitation in the substrate. Herein, we perform the first tip-enhanced Raman excitation spectroscopy (TERES) experiment and use the results, both in ambient and aqueous media, in combination with electrodynamics simulations, to explore the plasmonic response of a Au tip-Au substrate composite system. The gap-mode plasmon features a wide spectral window corresponding to a host of tip plasmon modes interacting with the plasmonic substrate. Simulations of the electric field confinement demonstrate that optimal spatial resolution is achieved when a hybrid plasmon mode that combines a multipolar tip plasmon and a substrate SPP is excited. Nevertheless, a wide spectral window over 1000 nm is available for exciting the tip plasmon with high spatial resolution, which enables the simultaneous resonant detection of different molecular species. This window is robust as a function of tip-substrate distance and tip radius of curvature, indicating that many choices of tips will work, but it is restricted to wavelengths longer than ∼600 nm for the Au tip-Au substrate combination. Other combinations, such as Ag tip-Ag substrate, can access wavelengths as low as 350 nm.

Experimental Determination of Magnetic Anisotropy in Exchange-Bias Dysprosium Metallocene Single-Molecule Magnets.

We investigate a family of dinuclear dysprosium metallocene single-molecule magnets (SMMs) bridged by methyl and halogen groups [Cp'2 Dy(µ-X)]2 (Cp'=cyclopentadienyltrimethylsilane anion; 1: X=CH3 - ; 2: X=Cl- ; 3: X=Br- ; 4: X=I- ). For the first time, the magnetic easy axes of dysprosium metallocene SMMs are experimentally determined, confirming that the orientation of them are perpendicular to the equatorial plane which is made up of dysprosium and bridging atoms. The orientation of the magnetic easy axis for 1 deviates from the normal direction (by 10.3°) due to the stronger equatorial interactions between DyIII and methyl groups. Moreover, its magnetic axes show a temperature-dependent shifting, which is caused by the competition between exchange interactions and Zeeman interactions. Studies of fluorescence and specific heat as well as ab initio calculations reveal the significant influences of the bridging ligands on their low-lying exchange-based energy levels and, consequently, low-temperature magnetic properties.

Two-Coordinate Co(II) Imido Complexes as Outstanding Single-Molecule Magnets.

The pursuit of single-molecule magnets (SMMs) with better performance urges new molecular design that can endow SMMs larger magnetic anisotropy. Here we report that two-coordinate cobalt imido complexes featuring highly covalent Co═N cores exhibit slow relaxation of magnetization under zero direct-current field with a high effective relaxation barrier up to 413 cm-1, a new record for transition metal based SMMs. Two theoretical models were carried out to investigate the anisotropy of these complexes: single-ion model and Co-N coupling model. The former indicates that the pseudo linear ligand field helps to preserve the first-order orbital momentum, while the latter suggests that the strong ferromagnetic interaction between Co and N makes the [CoN]+ fragment a pseudo single paramagnetic ion, and that the excellent performance of these cobalt imido SMMs is attributed to the inherent large magnetic anisotropy of the [CoN]+ core with |MJ = ± 7/2⟩ ground Kramers doublet.

STING Agonist Delivered by Neutrophil Membrane-Coated Gold Nanoparticles Exerts Synergistic Tumor Inhibition with Radiotherapy.

Radiotherapy (RT) is one of the major treatments for cancers and a promising initiator of immune response. Gold nanoparticles are a promising radiosensitizer. In this study, we sought to optimize the drug delivery efficiency of gold nanoparticles and explore their function in delivering stimulator of interferon genes (STING) agonists with or without RT. Gold nanoparticles covalent to MSA-2 (MSA-Au) were mixed with cRGD-modified neutrophil membranes to obtain M-Au@RGD-NM. We explored the treatment efficiency of M-Au@RGD-NM combined with RT. Immune cell regulation and STING pathway activation were detected. We successfully prepared M-Au@RGD-NM with significant tumor suppression by induction of ROS and the resulting DNA damage. In vivo dynamic imaging showed that M-Au@RGD-NM was mainly targeted to radiated tumors. Tumor-bearing mice showed significant tumor inhibition following a combination therapy. M-Au@RGD-NM significantly activated the STING pathway and regulated the whole-body immune response. Locally radiated tumors showed dendritic cells mature, CD8+ T cells upregulation, and M1 polarization, with systematic immune response demonstrated by CD8+ T cell infiltration in abscopal tumors. In this study, we synthesized M-Au@RGD-NM loading MSA-2. Following characterization, we found that RT-based M-Au@RGD-NM treatment achieved good antitumor effects, tumor RT enhancement, and induction of an immune response via STING activation.

Inhibition of DPAGT1 suppresses HER2 shedding and trastuzumab resistance in human breast cancer.

Human epidermal growth factor receptor 2-targeted (HER2-targeted) therapy is the mainstay of treatment for HER2+ breast cancer. However, the proteolytic cleavage of HER2, or HER2 shedding, induces the release of the target epitope at the ectodomain (ECD) and the generation of a constitutively active intracellular fragment (p95HER2), impeding the effectiveness of anti-HER2 therapy. Therefore, identifying key regulators in HER2 shedding might provide promising targetable vulnerabilities against resistance. In the current study, we found that upregulation of dolichyl-phosphate N-acetylglucosaminyltransferase (DPAGT1) sustained high-level HER2 shedding to confer trastuzumab resistance, which was associated with poor clinical outcomes. Upon trastuzumab treatment, the membrane-bound DPAGT1 protein was endocytosed via the caveolae pathway and retrogradely transported to the ER, where DPAGT1 induced N-glycosylation of the sheddase - ADAM metallopeptidase domain 10 (ADAM10) - to ensure its expression, maturation, and activation. N-glycosylation of ADAM10 at N267 protected itself from ER-associated protein degradation and was essential for DPAGT1-mediated HER2 shedding and trastuzumab resistance. Importantly, inhibition of DPAGT1 with tunicamycin acted synergistically with trastuzumab treatment to block HER2 signaling and reverse resistance. These findings reveal a prominent mechanism for HER2 shedding and suggest that targeting DPAGT1 might be a promising strategy against trastuzumab-resistant breast cancer.

BAG2 drives chemoresistance of breast cancer by exacerbating mutant p53 aggregate.

Rationale: Chemoresistance is a major challenge in the clinical management of patients with breast cancer. Mutant p53 proteins tend to form aggregates that promote tumorigenesis in cancers. We here aimed to explore the mechanism for the generation of mutant p53 aggregates in breast cancer and assess its role in inducing chemoresistance. Methods: Expression of BCL2-associated athanogene 2 (BAG2) was evaluated by qRT-PCR, western blotting, and immunohistochemistry in breast cancer patient specimens. The significance of BAG2 expression in prognosis was assessed by Kaplan-Meier survival analysis and the Cox regression model. The roles of BAG2 in facilitating the formation of mutant p53 aggregates were analyzed by co-immunoprecipitation, immunofluorescence, and semi-denaturing detergent-agarose gel electrophoresis assays. The effects of BAG2 on the chemoresistance of breast cancer were demonstrated by cell function assays and mice tumor models. Results: In the present study, we found that BAG2 was significantly upregulated in relapse breast cancer patient tissues and high BAG2 was associated with a worse prognosis. BAG2 localized in mutant p53 aggregates and interacted with misfolded p53 mutants. BAG2 exacerbated the formation of the aggregates and recruited HSP90 to promote the propagation and maintenance of the aggregates. Consequently, BAG2-mediated mutant p53 aggregation inhibited the mitochondrial apoptosis pathway, leading to chemoresistance in breast cancer. Importantly, silencing of BAG2 or pharmacological targeting of HSP90 substantially reduced the aggregates and increased the sensitivity of chemotherapy in breast cancer. Conclusion: These findings reveal a significant role of BAG2 in the chemoresistance of breast cancer via exacerbating mutant p53 aggregates and suggest that BAG2 may serve as a potential therapeutic target for breast cancer patients with drug resistance.

HMMR promotes peritoneal implantation of gastric cancer by increasing cell-cell interactions.

BACKGROUND: Distant metastasis is the prominent factor for cancer-induced death of gastric cancer in which peritoneum is one of the dominating targets of gastric cancer metastasis. However, there is still a lack of effective predictive indicators and treatment methods for gastric cancer patients with peritoneal metastasis. METHODS: A clustering assay was used to investigate the cell aggregates formation ability. While the soft agar assay and anoikis assay were performed to detect the anchorage-independent growth and anoikis-resistant ability respectively. Luciferase activity assay, western blotting and immunofluorescence were used to explore the effect of HMMR on AKT signaling activity. The peritoneal implantation model was examined to explore the role of HMMR in vivo. RESULTS: Silencing of HMMR expression markedly reduced the peritoneal metastasis of gastric cancer cells through reducing cell-cell interactions. Mechanistically, HA-HMMR could activate Akt signaling, thus succeeding in distant colonization and metastatic outgrowth. Importantly, inducible depletion of HMMR significantly abrogates peritoneal implantation of gastric cancer in vitro and in vivo. CONCLUSION: Our study highlights that HMMR promotes peritoneal implantation of gastric cancer. A better understanding of HMMR's functions and mechanism might provide a novel therapeutic target and prognostic marker for metastatic gastric cancer.