LRG1 promotes metastatic colorectal cancer growth through HER3 signaling.

BACKGROUND AND AIMS: Therapy failure in patients with metastatic colorectal cancer (mCRC, ∼80% occur in the liver) remains an overarching challenge. Preclinical studies demonstrated that HER3 promotes CRC cell survival, but therapies blocking the neuregulin-induced canonical HER3 signaling have made little impact in the clinic. Recent studies suggest that the liver microenvironment promotes CRC growth by activating HER3 in a neuregulin-independent fashion, thus elucidation of these mechanisms may reveal new strategies for treating patients with mCRC. METHODS: Patient-derived primary liver endothelial cells (ECs) were used to interrogate EC-CRC crosstalk. We conducted proteomic analysis to identify EC-secreted factor(s) that triggers non-canonical HER3 activation in CRC, and determined the subsequent effects on mCRC using diverse murine mCRC models. In vitro studies with genetic and pharmacological interventions were used to map the non-canonical HER3 pathway. RESULTS: We demonstrated that EC-secreted leucine-rich alpha-2-glycoprotein 1 (LRG1) directly binds and activates HER3 and promotes CRC growth distinct from neuregulin, the canonical HER3 ligand. Blocking host-derived LRG1 by gene knockout or a neutralizing antibody impaired mCRC outgrowth in the liver and prolonged mouse survival. We identified protein synthesis activated by the PI3K-PDK1-RSK-eIF4B axis as the biologically relevant signaling cascade downstream of the LRG1-HER3 interaction, which was not blocked by conventional HER3-specific antibodies that failed in prior clinical trials. CONCLUSIONS: LRG1 is a novel HER3 ligand and mediates liver-mCRC crosstalk. The LRG1-HER3 signaling axis is distinct from canonical HER3 signaling and represents a new therapeutic opportunity to treat patients with mCRC, and potentially other types of liver metastases.

Neutrophil extracellular traps induced by chemotherapy inhibit tumor growth in murine models of colorectal cancer.

Neutrophil extracellular traps (NETs), a web-like structure of cytosolic and granule proteins assembled on decondensed chromatin, kill pathogens and cause tissue damage in diseases. Whether NETs can kill cancer cells is unexplored. Here, we report that a combination of glutaminase inhibitor CB-839 and 5-FU inhibited the growth of PIK3CA-mutant colorectal cancers (CRCs) in xenograft, syngeneic, and genetically engineered mouse models in part through NETs. Disruption of NETs by either DNase I treatment or depletion of neutrophils in CRCs attenuated the efficacy of the drug combination. Moreover, NETs were present in tumor biopsies from patients treated with the drug combination in a phase II clinical trial. Increased NET levels in tumors were associated with longer progression-free survival. Mechanistically, the drug combination induced the expression of IL-8 preferentially in PIK3CA-mutant CRCs to attract neutrophils into the tumors. Further, the drug combination increased the levels of ROS in neutrophils, thereby inducing NETs. Cathepsin G (CTSG), a serine protease localized in NETs, entered CRC cells through the RAGE cell surface protein. The internalized CTSG cleaved 14-3-3 proteins, released BAX, and triggered apoptosis in CRC cells. Thus, our studies illuminate a previously unrecognized mechanism by which chemotherapy-induced NETs kill cancer cells.

PD-L1 expression is regulated by ATP-binding of the ERBB3 pseudokinase domain.

How PD-L1 expression is regulated in cancer is poorly understood. Here, we report that the ATP-binding activity of ERBB3 pseudokinase regulates PD-L1 gene expression in colorectal cancers (CRCs). ERBB3 is one of the four members of the EGF receptor family, all with protein tyrosine kinase domains. ERBB3 is a pseudokinase with a high binding affinity to ATP. We showed that ERBB3 ATP-binding inactivation mutant reduces tumorigenicity in genetically engineered mouse models and impairs xenograft tumor growth of CRC cell lines. The ERBB3 ATP-binding mutant cells dramatically reduce IFN-γ-induced PD-L1 expression. Mechanistically, ERBB3 regulates IFN-γ-induced PD-L1 expression through the IRS1-PI3K-PDK1-RSK-CREB signaling axis. CREB is the transcription factor that regulates PD-L1 gene expression in CRC cells. Knockin of a tumor-derived ERBB3 mutation located in the kinase domain sensitizes mouse colon cancers to anti-PD1 antibody therapy, suggesting that ERBB3 mutations could be predictive biomarkers for tumors amenable to immune checkpoint therapy.

Nuclear translocation of p85ß promotes tumorigenesis of PIK3CA helical domain mutant cancer.

PI3Ks consist of p110 catalytic subunits and p85 regulatory subunits. PIK3CA, encoding p110α, is frequently mutated in human cancers. Most PIK3CA mutations are clustered in the helical domain or the kinase domain. Here, we report that p85ß disassociates from p110α helical domain mutant protein and translocates into the nucleus through a nuclear localization sequence (NLS). Nuclear p85ß recruits deubiquitinase USP7 to stabilize EZH1 and EZH2 and enhances H3K27 trimethylation. Knockout of p85ß or p85ß NLS mutant reduces the growth of tumors harboring a PIK3CA helical domain mutation. Our studies illuminate a novel mechanism by which PIK3CA helical domain mutations exert their oncogenic function. Finally, a combination of Alpelisib, a p110α-specific inhibitor, and an EZH inhibitor, Tazemetostat, induces regression of xenograft tumors harboring a PIK3CA helical domain mutation, but not tumors with either a WT PIK3CA or a PIK3CA kinase domain mutation, suggesting that the drug combination could be an effective therapeutic approach for PIK3CA helical domain mutant tumors.

Transcriptome Analysis Identifies SenZfp536, a Sense LncRNA that Suppresses Self-renewal of Cortical Neural Progenitors.

Long non-coding RNAs (lncRNAs) regulate transcription to control development and homeostasis in a variety of tissues and organs. However, their roles in the development of the cerebral cortex have not been well elucidated. Here, a bioinformatics pipeline was applied to delineate the dynamic expression and potential cis-regulating effects of mouse lncRNAs using transcriptome data from 8 embryonic time points and sub-regions of the developing cerebral cortex. We further characterized a sense lncRNA, SenZfp536, which is transcribed downstream of and partially overlaps with the protein-coding gene Zfp536. Both SenZfp536 and Zfp536 were predominantly expressed in the proliferative zone of the developing cortex. Zfp536 was cis-regulated by SenZfp536, which facilitates looping between the promoter of Zfp536 and the genomic region that transcribes SenZfp536. Surprisingly, knocking down or activating the expression of SenZfp536 increased or compromised the proliferation of cortical neural progenitor cells (NPCs), respectively. Finally, overexpressing Zfp536 in cortical NPCs reversed the enhanced proliferation of cortical NPCs caused by SenZfp536 knockdown. The study deepens our understanding of how lncRNAs regulate the propagation of cortical NPCs through cis-regulatory mechanisms.

Long non-coding RNA LncKdm2b regulates cortical neuronal differentiation by cis-activating Kdm2b.

The mechanisms underlying spatial and temporal control of cortical neurogenesis of the brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed during early differentiation of cortical projection neurons. Interestingly, Kdm2b's transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating function and facilitates a permissive chromatin environment at the Kdm2b's promoter by associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of cortical projection neurons. These observations unveiled a lncRNA-dependent machinery in regulating cortical neuronal differentiation.

Paired related homeobox 1 transactivates dopamine D2 receptor to maintain propagation and tumorigenicity of glioma-initiating cells.

Glioblastoma multiforme (GBM) is a highly invasive brain tumor with limited therapeutic means and poor prognosis. Recent studies indicate that glioma-initiating cells/glioma stem cells (GICs/GSCs) may be responsible for tumor initiation, infiltration, and recurrence. GICs could aberrantly employ molecular machinery balancing self-renewal and differentiation of embryonic neural precursors. Here, we find that paired related homeobox 1 (PRRX1), a homeodomain transcription factor that was previously reported to control skeletal development, is expressed in cortical neural progenitors and is required for their self-renewal and proper differentiation. Further, PRRX1 is overrepresented in glioma samples and labels GICs. Glioma cells and GICs depleted with PRRX1 could not propagate in vitro or form tumors in the xenograft mouse model. The GIC self-renewal function regulated by PRRX1 is mediated by dopamine D2 receptor (DRD2). PRRX1 directly binds to the DRD2 promoter and transactivates its expression in GICs. Blockage of the DRD2 signaling hampers GIC self-renewal, whereas its overexpression restores the propagating and tumorigenic potential of PRRX1-depleted GICs. Finally, PRRX1 potentiates GICs via DRD2-mediated extracellular signal-related kinase (ERK) and AKT activation. Thus, our study suggests that therapeutic targeting the PRRX1-DRD2-ERK/AKT axis in GICs is a promising strategy for treating GBMs.

HMGA2 sustains self-renewal and invasiveness of glioma-initiating cells.

Glioblastoma multiforme (GBM) is the most common type of brain tumors with dismal outcomes. The mesenchymal phenotype is the hallmark of tumor aggressiveness in GBMs. Perivascular smooth muscle cells (pericytes) are essential in homeostasis of normal and glioma tissues. Here we found HMGA2, an architectural transcription factor that promotes mesenchymal phenotypes in a number of solid tumors, is highly expressed in mesenchymal subtype of GBMs and labels both glioma pericytes and glioma-initiating cells (GICs). Accordingly, depletion of HMGA2 in GICs resulted in compromised self-renewal and tumorigenic capability, as well as undermined mesenchymal or pericyte differentiation. We further showed HMGA2 allows expressions of FOXM1 and PLAU to maintain GIC propagation, gliomagenesis and aggressiveness both in vitro and in vivo. Therefore, suppressing HMGA2-mediated GIC self-renewal and invasiveness might be a promising means to treat GBMs.

Novel function of PITH domain-containing 1 as an activator of internal ribosomal entry site to enhance RUNX1 expression and promote megakaryocyte differentiation.

INTRODUCTION: Altered gene expression coincides with leukemia development and may affect distinct features of leukemic cells. PITHD1 was significantly downregulated in leukemia and upregulated upon PMA induction in K562 cells undergoing megakaryocyte differentiation. We aimed to study the function of PITHD1 in megakaryocyte differentiation. MATERIALS AND METHODS: K562 cells and fetal liver cells were used for either overexpression or downregulation of PITHD1 by retroviral or lentiviral transduction. FACS was used to detect the expression of CD41 and CD42 to measure megakaryocyte differentiation in these cells. Western blot and quantitative RT-PCR were used to measure gene expression. Dual luciferase assay was used to detect promoter or internal ribosomal entry site (IRES) activity. RESULTS: Ectopic expression of PITHD1 promoted megakaryocyte differentiation and increased RUNX1 expression while PITHD1 knockdown showed an opposite phenotype. Furthermore, PITHD1 efficiently induced endogenous RUNX1 expression and restored megakaryocyte differentiation suppressed by a dominant negative form of RUNX1. PITHD1 regulated RUNX1 expression at least through two distinct mechanisms: increasing transcription activity of proximal promoter and enhancing translation activity of an IRES element in exon 3. Finally, we confirmed the function of PITHD1 in regulating RUNX1 expression and megakaryopoiesis in mouse fetal liver cells. CONCLUSION AND SIGNIFICANCE: PITHD1 was a novel activator of IRES and enhanced RUNX1 expression that subsequently promoted megakaryocyte differentiation. Our findings shed light on understanding the mechanisms underlying megakaryopoiesis or leukemogenesis.

Transcriptome and proteome exploration to provide a resource for the study of Agrocybe aegerita.

BACKGROUND: Agrocybe aegerita, the black poplar mushroom, has been highly valued as a functional food for its medicinal and nutritional benefits. Several bioactive extracts from A. aegerita have been found to exhibit antitumor and antioxidant activities. However, limited genetic resources for A. aegerita have hindered exploration of this species. METHODOLOGY/PRINCIPAL FINDINGS: To facilitate the research on A. aegerita, we established a deep survey of the transcriptome and proteome of this mushroom. We applied high-throughput sequencing technology (Illumina) to sequence A. aegerita transcriptomes from mycelium and fruiting body. The raw clean reads were de novo assembled into a total of 36,134 expressed sequences tags (ESTs) with an average length of 663 bp. These ESTs were annotated and classified according to Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways. Gene expression profile analysis showed that 18,474 ESTs were differentially expressed, with 10,131 up-regulated in mycelium and 8,343 up-regulated in fruiting body. Putative genes involved in polysaccharide and steroid biosynthesis were identified from A. aegerita transcriptome, and these genes were differentially expressed at the two stages of A. aegerita. Based on one-dimensional gel electrophoresis (1-DGE) coupled with electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS), we identified a total of 309 non-redundant proteins. And many metabolic enzymes involved in glycolysis were identified in the protein database. CONCLUSIONS/SIGNIFICANCE: This is the first study on transcriptome and proteome analyses of A. aegerita. The data in this study serve as a resource of A. aegerita transcripts and proteins, and offer clues to the applications of this mushroom in nutrition, pharmacy and industry.

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine.

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo.