ASPM stabilizes the NOTCH intracellular domain 1 and promotes oncogenesis by blocking FBXW7 binding in hepatocellular carcinoma cells.

Notch signaling is aberrantly activated in approximately 30% of hepatocellular carcinoma (HCC), significantly contributing to tumorigenesis and disease progression. Expression of the major Notch receptor, NOTCH1, is upregulated in HCC cells and correlates with advanced disease stages, although the molecular mechanisms underlying its overexpression remain unclear. Here, we report that expression of the intracellular domain of NOTCH1 (NICD1) is upregulated in HCC cells due to antagonism between the E3-ubiquitin ligase F-box/WD repeat-containing protein 7 (FBXW7) and the large scaffold protein abnormal spindle-like microcephaly-associated protein (ASPM) isoform 1 (ASPM-i1). Mechanistically, FBXW7-mediated polyubiquitination and the subsequent proteasomal degradation of NICD1 are hampered by the interaction of NICD1 with ASPM-i1, thereby stabilizing NICD1 and rendering HCC cells responsive to stimulation by Notch ligands. Consistently, downregulating ASPM-i1 expression reduced the protein abundance of NICD1 but not its FBXW7-binding-deficient mutant. Reinforcing the oncogenic function of this regulatory module, the forced expression of NICD1 significantly restored the tumorigenic potential of ASPM-i1-deficient HCC cells. Echoing these findings, NICD1 was found to be strongly co-expressed with ASPM-i1 in cancer cells in human HCC tissues (P < 0.001). In conclusion, our study identifies a novel Notch signaling regulatory mechanism mediated by protein-protein interaction between NICD1, FBXW7, and ASPM-i1 in HCC cells, representing a targetable vulnerability in human HCC.

ASPM Activates Hedgehog and Wnt Signaling to Promote Small Cell Lung Cancer Stemness and Progression.

Small cell lung cancer (SCLC) is among the most aggressive and lethal human malignancies. Most patients with SCLC who initially respond to chemotherapy develop disease relapse. Therefore, there is a pressing need to identify novel driver mechanisms of SCLC progression to unlock treatment strategies to improve patient prognosis. SCLC cells comprise subsets of cells possessing progenitor or stem cell properties, while the underlying regulatory pathways remain elusive. Here, we identified the isoform 1 of the neurogenesis-associated protein ASPM (ASPM-I1) as a prominently upregulated stemness-associated gene during the self-renewal of SCLC cells. The expression of ASPM-I1 was found to be upregulated in SCLC cells and tissues, correlated with poor patient prognosis, and indispensable for SCLC stemness and tumorigenesis. A reporter array screening identified multiple developmental signaling pathways, including Hedgehog (Hh) and Wnt pathways, whose activity in SCLC cells depended upon ASPM-I1 expression. Mechanistically, ASPM-I1 stabilized the Hh transcriptional factor GLI1 at the protein level through a unique exon-18-encoded region by competing with the E3 ligases ß-TrCP and CUL3. In parallel, ASPM-I1 sustains the transcription of the Hh pathway transmembrane regulator SMO through the Wnt-DVL3-ß-catenin signaling axis. Functional studies verified that the ASPM-I1-regulated Hh and Wnt activities significantly contributed to SCLC aggressiveness in vivo. Consistently, the expression of ASPM-I1 positively correlated with GLI1 and stemness markers in SCLC tissues. This study illuminates an ASPM-I1-mediated regulatory module that drives tumor stemness and progression in SCLC, providing an exploitable diagnostic and therapeutic target. SIGNIFICANCE: ASPM promotes SCLC stemness and aggressiveness by stabilizing the expression of GLI1, DVL3, and SMO, representing a novel regulatory hub of Hh and Wnt signaling and targetable vulnerability.

Corrigendum: A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

[This corrects the article DOI: 10.3389/fonc.2021.638311.].

A Novel Invadopodia-Specific Marker for Invasive and Pro-Metastatic Cancer Stem Cells.

INTRODUCTION: Stem-like cancer cells or cancer stem cells (CSCs) may comprise a phenotypically and functionally heterogeneous subset of cells, whereas the molecular markers reflecting this CSC hierarchy remain elusive. The glycolytic enzyme alpha-enolase (ENO1) present on the surface of malignant tumor cells has been identified as a metastasis-promoting factor through its function of activating plasminogen. The expression pattern of surface ENO1 (sENO1) concerning cell-to-cell or CSC heterogeneity and its functional roles await further investigation. METHODS: The cell-to-cell expression heterogeneity of sENO1 was profiled in malignant cells from different types of cancers using flow cytometry. The subcellular localization of sENO1 and its functional roles in the invadopodia formation and cancer cell invasiveness were investigated using a series of imaging, molecular, and in vitro and in vivo functional studies. RESULTS: We showed here that ENO1 is specifically localized to the invadopodial surface of a significant subset (11.1%-63.9%) of CSCs in human gastric and prostate adenocarcinomas. sENO1+ CSCs have stronger mesenchymal properties than their sENO1- counterparts. The subsequent functional studies confirmed the remarkable pro-invasive and pro-metastatic capacities of sENO1+ CSCs. Mechanistically, inhibiting the surface localization of ENO1 by downregulating caveolin-1 expression compromised invadopodia biogenesis, proteolysis, and CSC invasiveness. CONCLUSIONS: Our study identified the specific expression of ENO1 on the invadopodial surface of a subset of highly invasive and pro-metastatic CSCs. sENO1 may provide a diagnostically and/or therapeutically exploitable target to improve the outcome of patients with aggressive and metastatic cancers.

A multi-mode Wnt- and stemness-regulatory module dictated by FOXM1 and ASPM isoform I in gastric cancer.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer mortality globally and a molecularly heterogeneous disease. Identifying the driver pathways in GC progression is crucial to improving the clinical outcome. Recent studies identified ASPM (abnormal spindle-like microcephaly-associated) and FOXM1 (Forkhead box protein M1) as novel Wnt and cancer stem cell (CSC) regulators; their pathogenetic roles and potential crosstalks in GC remain unclarified. METHODS: The expression patterns of ASPM isoforms and FOXM1 were profiled in normal gastric epithelial and GC tissues. The functional roles of ASPM and FOXM1 in Wnt activity, cancer stemness and GC progression, and the underlying signaling processes were investigated. RESULTS: Approximately one third of GC cells upregulate the expression of ASPM isoform I (ASPMiI) in their cytoplasm; the tumors with a high ASPMiI positive score (≥ 10%) are associated with a poor prognosis of the patients. Mechanistically, the molecular interplay among FOXM1, ASPMiI and DVL3 was found to converge on ß-catenin to control the Wnt activity and the stemness property of GC cells. This multi-mode Wnt-regulatory module serves to reinforce Wnt signals in CSCs by transcriptional regulation (FOXM1-ASPM), protein-protein interactions (ASPMiI-DVL3-ß-catenin), and nuclear translocation (FOXM1-ß-catenin). CONCLUSIONS: This study illuminates a novel Wnt- and stemness-regulatory mechanism in GC cells and identifies a novel subset of FOXM1highASPMiIhigh GC with potential to guide Wnt- and stemness-related diagnostics and therapies.

An intranasally delivered peptide drug ameliorates cognitive decline in Alzheimer transgenic mice.

Alzheimer's disease (AD) is the most common neurodegenerative disease. Imbalance between the production and clearance of amyloid ß (Aß) peptides is considered to be the primary mechanism of AD pathogenesis. This amyloid hypothesis is supported by the recent success of the human anti-amyloid antibody aducanumab, in clearing plaque and slowing clinical impairment in prodromal or mild patients in a phase Ib trial. Here, a peptide combining polyarginines (polyR) (for charge repulsion) and a segment derived from the core region of Aß amyloid (for sequence recognition) was designed. The efficacy of the designed peptide, R8-Aß(25-35), on amyloid reduction and the improvement of cognitive functions were evaluated using APP/PS1 double transgenic mice. Daily intranasal administration of PEI-conjugated R8-Aß(25-35) peptide significantly reduced Aß amyloid accumulation and ameliorated the memory deficits of the transgenic mice. Intranasal administration is a feasible route for peptide delivery. The modular design combining polyR and aggregate-forming segments produced a desirable therapeutic effect and could be easily adopted to design therapeutic peptides for other proteinaceous aggregate-associated diseases.

Intranasal Administration of a Polyethylenimine-Conjugated Scavenger Peptide Reduces Amyloid-ß Accumulation in a Mouse Model of Alzheimer's Disease.

Amyloid-ß (Aß) aggregation in the brain plays a central and initiatory role in pathogenesis and/or progression of Alzheimer's disease (AD). Inhibiting Aß aggregation is a potential strategy in the prevention of AD. A scavenger peptide, V24P(10-40), designed to decrease Aß accumulation in the brain, was conjugated to polyethylenimine (PEI) and tested as a preventive/therapeutic strategy for AD in this study. This PEI-conjugated V24P(10-40) peptide was delivered intranasally, as nasal drops, to four-month-old APP/PS1 double transgenic mice for four or eight months. Compared with control values, peptide treatment for four months significantly reduced the amount of GdnHCl-extracted Aß40 and Aß42 in the mice's hippocampus and cortex. After treatment for eight months, amyloid load, as quantified by Pittsburgh compound B microPET imaging, was significantly decreased in the mice's hippocampus, cortex, amygdala, and olfactory bulb. Our data suggest that this intranasally delivered scavenger peptide is effective in decreasing Aß accumulation in the brain of AD transgenic mice. Nasal application of peptide drops is easy to use and could be further developed to prevent and treat AD.

How do amino acid substitutions affect the amyloidogenic properties and seeding efficiency of prion peptides.

The amino acid sequences in the amyloidogenic region (amino acids 108-144) of several mammalian prion proteins were compared and variations were found to occur at residues 109 (M or L), 112 (M or V), 129 (M, V, or L), 135 (N or S), 138 (M, L, or I), 139 (M or I), and 143 (N or S). Using the bovine PrP peptide (residues 108-144 based on the numbering of the human prion protein sequence) as a control peptide, several peptides with one amino acid differing from that of the bovine PrP peptide at residues 109, 112, 135, 138, 139, or 143 and several mammalian PrP peptides were synthesized, and the effects of these amino acid substitutions on the amyloidogenic properties of these peptides were compared and discussed on the basis of the chemical and structural properties of amino acids. Our results showed that the V112M substitution accelerated nucleation of amyloidogenesis, while the N143S and I139M substitutions retarded nucleation. These effects tended to cancel each other out when two substitutions with opposite effects were present on the same peptide. Moreover, acceleration or inhibition of nucleation was not necessarily correlated with effect on seeding efficiency. Using amyloid fibrils prepared from the bovine PrP peptide as seeds, the seeding efficiency for the monomer peptides with the M129L, S135N, N143S, or I139M substitution was decreased compared to that for bPrP peptide. Of all the mammalian peptides used in this study, the dog, mule deer, and pig PrP peptides had the lowest seeding efficiencies.

Leu138 in bovine prion peptide fibrils is involved in seeding discrimination related to codon 129 M/V polymorphism in the prion peptide seeding experiment.

The risk of acquiring variant Creutzfeldt-Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt-Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed 'seed-titration method' and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the Met→Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a 'surface competition hypothesis' to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.

A highly sensitive peptide substrate for detecting two Aß-degrading enzymes: neprilysin and insulin-degrading enzyme.

Neprilysin has been singled out as the most promising candidate for use in the degradation of Abeta as a therapy for Alzheimer's disease. In this study, a quenched fluorogenic peptide substrate containing the first seven residues of the Abeta peptide plus a C-terminal Cysteine residue was synthesized to detect neprilysin activity. A fluorophore was attached to the C-terminal Cysteine and its fluorescence was quenched by a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, fluorescence was produced and proved to be a sensitive detection system for endopeptidase activity. Our results showed that this assay system was extremely sensitive to neprilysin and insulin-degrading enzyme, but insensitive, or much less sensitive, to other Abeta-degrading enzymes. As low as 0.1 nM of neprilysin and 0.2 nM of insulin-degrading enzyme can be detected.

Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.

Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions.

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.

A new amyloid-like beta-aggregate with amyloid characteristics, except fibril morphology.

Amyloid plaques, formed from amyloid beta (Abeta) peptides (mainly Abeta40 or Abeta42), are one of the most important pathological characteristics of Alzheimer's disease. Here, a single D-form proline substitution in the 40-amino-acid Abeta40 peptide can totally change the aggregation behavior of this peptide. The residue immediately preceding each glycine in Abeta40 (S8, V24, I32, and V36) was individually replaced by D-form proline ((D)Pro). The resulting (D)P-G sequence (the (D)Pro residue and the following Gly residue) was designed as a "structural clip" to force the formation of a bend in the peptide, as this sequence has been reported to be a strong promoter of beta-hairpin formation. The mutant peptide with Val24-to-(D)Pro substitution, named V24P, formed a new amyloid-like beta-aggregate at high peptide concentration. The aggregate has most of the characteristics of amyloid fibrils, except fibril morphology. Moreover, the mutant peptide V24P, when mixed with Abeta40, can attenuate the cytotoxicity of Abeta40.