Trajectory of immune evasion and cancer progression in hepatocellular carcinoma.

Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.

Fluid shear stress activates YAP to promote epithelial-mesenchymal transition in hepatocellular carcinoma.

Epithelial-mesenchymal transition (EMT) mediated by fluid shear stress (FSS) in the tumor microenvironment plays an important role in driving metastasis of the malignant tumor. As a mechanotransducer, Yes-associated protein (YAP) is known to translocate into the nucleus to initiate transcription of genes involved in cell proliferation upon extracellular biophysical stimuli. Here, we showed that FSS facilitated cytoskeleton rearrangement in hepatocellular carcinoma cells, which led to the release of YAP from its binding partner, integrin ß subunit, in the cytomembrane. Moreover, we found that upregulation of guanine nucleotide exchange factor (GEF)-H1, a microtubule-associated Rho GEF, is a critical step in the FSS-induced translocation of YAP. Nuclear YAP activated the expression of the EMT-regulating transcription factor SNAI1, but suppressed the expression of N6-methyladenosine (m6 A) modulators; together, this promoted the expression of EMT-related genes. We also observed that FSS-treated HepG2 cells showed markedly increased tumorigenesis and metastasis in vivo. Collectively, our findings unravel the underlying molecular processes by which FSS induces translocation of YAP from the cytomembrane to the nucleus, contributes to EMT and enhances metastasis in hepatocellular carcinoma.

Construction of developmental lineage relationships in the mouse mammary gland by single-cell RNA profiling.

The mammary epithelium comprises two primary cellular lineages, but the degree of heterogeneity within these compartments and their lineage relationships during development remain an open question. Here we report single-cell RNA profiling of mouse mammary epithelial cells spanning four developmental stages in the post-natal gland. Notably, the epithelium undergoes a large-scale shift in gene expression from a relatively homogeneous basal-like program in pre-puberty to distinct lineage-restricted programs in puberty. Interrogation of single-cell transcriptomes reveals different levels of diversity within the luminal and basal compartments, and identifies an early progenitor subset marked by CD55. Moreover, we uncover a luminal transit population and a rare mixed-lineage cluster amongst basal cells in the adult mammary gland. Together these findings point to a developmental hierarchy in which a basal-like gene expression program prevails in the early post-natal gland prior to the specification of distinct lineage signatures, and the presence of cellular intermediates that may serve as transit or lineage-primed cells.

The mammary stem cell hierarchy.

The mammary epithelium undergoes enormous morphogenetic changes during the lifespan of a mammal. The recent elucidation of an epithelial differentiation hierarchy in the mouse mammary gland through classical transplantation and clonogenic assays has pointed to the existence of multipotent mammary stem cells (MaSCs) and at least two distinct luminal progenitor types. Moreover, an analogous functional hierarchy has been defined in human breast tissue. The existence of slow cycling stem cells, both long- and short-term repopulating cells, and a unique fetal MaSC population, imply a complex stem cell compartment within the mammary gland. The recent discovery of unipotent stem-like cells from lineage tracing studies has added a further layer of complexity to the emerging differentiation hierarchy. Although the precise relationships between stem and progenitor cells have yet to be resolved, the epithelial hierarchy has provided an important framework for elucidating the roles of molecular regulators of mammary gland ontogeny and understanding potential cells of origin in breast cancer.

Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2.

The mammary epithelium is a dynamic, highly hormone-responsive tissue. To explore chromatin modifications underlying its lineage specification and hormone responsiveness, we determined genome-wide histone methylation profiles of mammary epithelial subpopulations in different states. The marked differences in H3K27 trimethylation between subpopulations in the adult gland suggest that epithelial cell-fate decisions are orchestrated by polycomb-complex-mediated repression. Remarkably, the mammary epigenome underwent highly specific changes in different hormonal contexts, with a profound change being observed in the global H3K27me3 map of luminal cells during pregnancy. We therefore examined the role of the key H3K27 methyltransferase Ezh2 in mammary physiology. Its expression and phosphorylation coincided with H3K27me3 modifications and peaked during pregnancy, driven in part by progesterone. Targeted deletion of Ezh2 impaired alveologenesis during pregnancy, preventing lactation, and drastically reduced stem/progenitor cell numbers. Taken together, these findings reveal that Ezh2 couples hormonal stimuli to epigenetic changes that underpin progenitor activity, lineage specificity, and alveolar expansion in the mammary gland.

TRIM39 regulates cell cycle progression and DNA damage responses via stabilizing p21.

The biological function of Tripartite Motif 39 (TRIM39) remains largely unknown. In this study, we report that TRIM39 regulates the steady-state levels of p21 and is a pivotal determinant of cell fate. Ablation of TRIM39 leads to destabilization of p21 and increased G1/S transition in unperturbed cells. Furthermore, DNA damage-induced p21 accumulation is completely abolished in cells with depleted TRIM39. As a result, silencing of TRIM39 abrogates the G2 checkpoint induced by genotoxic stress, leading to increased mitotic entry and, ultimately, apoptosis. Importantly, we show p21 is a crucial downstream effector of TRIM39 mediating G1/S transition and DNA damage-induced G2 arrest. Mechanistically, TRIM39 interacts with p21, which subsequently prevents Cdt2 from binding to p21, therefore blocking ubiquitylation and proteasomal degradation of p21 mediated by CRL4(Cdt2) E3 ligase. Strikingly, we found a significant correlation between p21 abundance and TRIM39 expression levels in human hepatocellular carcinoma samples. Our findings identify a causal role for TRIM39 in regulating cell cycle progression and the balance between cytostasis and apoptosis after DNA damage via stabilizing p21.

Effects of Cyclosporine A and Highly Expressed Bcl-2 on Apoptosis of HL-60 Cells Induced by EGTA.

Effects of mitochondrial permeability transition pore-specific inhibitor cyclosporine A (CsA) and highly expressed Bcl-2 on the apoptosis of HL-60 cells induced by EGTA were studied. Detection of apoptotic peak by flow cytometry, fluorescent microscope observation of chromatin condensation with double staining of PI and Hoechst33342 and DNA ladder analysis all demonstrated that CsA obviously enhanced the apoptosis of HL-60 cells induced by EGTA, while highly expressed Bcl-2 completely blocked it. It is revealed by mitochondrial membrane potential (deltapsi(m)) fluorescent probes rhodamine 123 and CMXRos that the deltapsi(m) decreased in the apoptosis of HL-60 cells induced by EGTA. CsA enhanced the decrease of deltapsi(m), but highly expressed Bcl-2 increased deltapsi(m) of HL-60 cells about 2-fold and completely blocked the decrease of deltapsi(m).

Relationship between Superoxide Anion Radical and Anion Transport Protein of Red Blood Cell Membrane.

By using a specific inhibitor of anion transport, DIDS, the dependence on band 3 anion transport protein of superoxide anion radical (O(-)(2).) flux across red blood cell (RBC) membrane into RBC was studied and the effect of superoxide anion radical on anion transport in RBC membrane was also discussed based on the hemolytic t(1/2) in an isotonic solution of NH(4)Cl. The results showed that 40 &mgr;mol/L DIDS could completely inhibit anion transport by band 3 protein in RBC membrane, but had no effect on flux into RBC, proving that the O(-)(2). transport into RBC across RBC membrane was independent of band 3 protein the interaction of O(-)(2). with specific SH-group of membrane protein lowered the anion permeability, and it could be reversed by DTT and mercaptoethanol.