Cardiovascular protection of YiyiFuzi powder and the potential mechanisms through modulating mitochondria-endoplasmic reticulum interactions.

Cardiovascular diseases (CVD) remain the leading cause of death worldwide and represent a major public health challenge. YiyiFuzi Powder (YYFZ), composed of Coicis semen and Fuzi, is a classical traditional Chinese medicine prescription from the Synopsis of Golden Chamber dating back to the Han Dynasty. Historically, YYFZ has been used to treat various CVD, rooted in Chinese therapeutic principles. Network pharmacology analysis indicated that YYFZ may exhibit direct or indirect effects on mitochondria-endoplasmic reticulum (ER) interactions. This review, focusing on the cardiovascular protective effects of Coicis semen and Fuzi, summarizes the potential mechanisms by which YYFZ acts on mitochondria and the ER. The underlying mechanisms are associated with regulating cardiovascular risk factors (such as blood lipids and glucose), impacting mitochondrial structure and function, modulating ER stress, inhibiting oxidative stress, suppressing inflammatory responses, regulating cellular apoptosis, and maintaining calcium ion balance. The involved pathways include, but were not limited to, upregulating the IGF-1/PI3K/AKT, cAMP/PKA, eNOS/NO/cGMP/SIRT1, SIRT1/PGC-1α, Klotho/SIRT1, OXPHOS/ATP, PPARα/PGC-1α/SIRT3, AMPK/JNK, PTEN/PI3K/AKT, ß2-AR/PI3K/AKT, and modified Q cycle signaling pathways. Meanwhile, the MCU, NF-κB, and JAK/STAT signaling pathways were downregulated. The PERK/eIF2α/ATF4/CHOP, PERK/SREBP-1c/FAS, IRE1, PINK1-dependent mitophagy, and AMPK/mTOR signaling pathways were bidirectionally regulated. High-quality experimental studies are needed to further elucidate the underlying mechanisms of YYFZ in CVD treatment.

Physics-driven structural docking and protein language models accelerate antibody screening and design for broad-spectrum antiviral therapy.

Therapeutic antibodies have become one of the most influential therapeutics in modern medicine to fight against infectious pathogens, cancer, and many other diseases. However, experimental screening for highly efficacious targeting antibodies is labor-intensive and of high cost, which is exacerbated by evolving antigen targets under selective pressure such as fast-mutating viral variants. As a proof-of-concept, we developed a machine learning-assisted antibody generation pipeline that greatly accelerates the screening and re-design of immunoglobulins G (IgGs) against a broad spectrum of SARS-CoV-2 coronavirus variant strains. These viruses infect human host cells via the viral spike protein binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). Using over 1300 IgG sequences derived from convalescent patient B cells that bind with spike's receptor binding domain (RBD), we first established protein structural docking models in assessing the RBD-IgG-ACE2 interaction interfaces and predicting the virus-neutralizing activity of each IgG with a confidence score. Additionally, employing Gaussian process regression (also known as Kriging) in a latent space of an antibody language model, we predicted the landscape of IgGs' activity profiles against individual coronaviral variants of concern. With functional analyses and experimental validations, we efficiently prioritized IgG candidates for neutralizing a broad spectrum of viral variants (wildtype, Delta, and Omicron) to prevent the infection of host cells in vitro and hACE2 transgenic mice in vivo. Furthermore, the computational analyses enabled rational redesigns of selective IgG clones with single amino acid substitutions at the RBD-binding interface to improve the IgG blockade efficacy for one of the severe, therapy-resistant strains - Delta (B.1.617). Our work expedites applications of artificial intelligence in antibody screening and re-design even in low-data regimes combining protein language models and Kriging for antibody sequence analysis, activity prediction, and efficacy improvement, in synergy with physics-driven protein docking models for antibody-antigen interface structure analyses and functional optimization.

PRL2 Phosphatase Promotes Oncogenic KIT Signaling in Leukemia Cells through Modulating CBL Phosphorylation.

Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.

Curcumin alleviates orofacial allodynia and improves cognitive impairment via regulating hippocampal synaptic plasticity in a mouse model of trigeminal neuralgia.

OBJECTIVE: Cognitive impairment, one of the most prevalent complications of trigeminal neuralgia, is troubling for patients and clinicians due to limited therapeutic options. Curcumin shows antinociception and neuroprotection pharmacologically, suggesting that it may have therapeutic effect on this complication. This study aimed to investigate whether curcumin alleviates orofacial allodynia and improves cognitive impairment by regulating hippocampal CA1 region synaptic plasticity in trigeminal neuralgia. METHODS: A mouse model of trigeminal neuralgia was established by partially transecting the infraorbital nerve (pT-ION). Curcumin was administered by gavage twice daily for 14 days. Nociceptive thresholds were measured using the von Frey and acetone test, and the cognitive functions were evaluated using the Morris water maze test. Dendritic spines and synaptic ultrastructures in the hippocampal CA1 area were observed by Golgi staining and transmission electron microscopy. RESULTS: Curcumin intervention increased the mechanical and cold pain thresholds of models. It decreased the escape latency and distance to the platform and increased the number of platform crossings and dwell time in the target quadrant of models, and improved spatial learning and memory deficits. Furthermore, it partially restored the disorder of the density and proportion of dendritic spines and the abnormal density and structure of synapses in the hippocampal CA1 region of models. CONCLUSION: Curcumin alleviates abnormal orofacial pain and cognitive impairment in pT-ION mice by a mechanism that may be related to the synaptic plasticity of hippocampal CA1, suggesting that curcumin is a potential strategy for repairing cognitive dysfunction under long-term neuropathic pain conditions.

Ubiquitin-specific peptidase 22 controls integrin-dependent cancer cell stemness and metastasis.

Integrins plays critical roles in connecting the extracellular matrix and actin skeleton for cell adhesion, migration, signal transduction, and gene transcription, which upregulation is involved in cancer stemness and metastasis. However, the molecular mechanisms underlying how integrins are upregulated in cancer stem cells (CSCs) remain as a biomedical mystery. Herein, we show that the death from cancer signature gene USP22 is essential to maintain the stemness of breast cancer cells through promoting the transcription of a group of integrin family members in particular integrin ß1 (ITGB1). Both genetic and pharmacological USP22 inhibition largely impaired breast cancer stem cell self-renewal and prevented their metastasis. Integrin ß1 reconstitution partially rescued USP22-null breast cancer stemness and their metastasis. At the molecular level, USP22 functions as a bona fide deubiquitinase to protect the proteasomal degradation of the forkhead box M1 (FoxM1), a transcription factor for tumoral ITGB1 gene transcription. Importantly unbiased analysis of the TCGA database revealed a strong positive correlation between the death from cancer signature gene ubiquitin-specific peptidase 22 (USP22) and ITGB1, both of which are critical for cancer stemness, in more than 90% of human cancer types, implying that USP22 functions as a key factor to maintain stemness for a broad spectrum of human cancer types possibly through regulating ITGB1. To support this notion, immunohistochemistry staining detected a positive correlation among USP22, FoxM1 and integrin ß1 in human breast cancers. Collectively, our study identifies the USP22-FoxM1-integrin ß1 signaling axis critical for cancer stemness and offers a potential target for antitumor therapy.

Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins. Dynamic hyposialylation in CTCs or deficiency of ST6GAL1 promotes cluster formation for metastatic seeding and enables cellular quiescence to evade paclitaxel treatment in breast cancer. Glycoproteomic analysis reveals newly identified protein substrates of ST6GAL1, such as adhesion or stemness markers PODXL, ICAM1, ECE1, ALCAM1, CD97, and CD44, contributing to CTC clustering (aggregation) and metastatic seeding. As a proof of concept, neutralizing antibodies against one newly identified contributor, PODXL, inhibit CTC cluster formation and lung metastasis associated with paclitaxel treatment for triple-negative breast cancer. SIGNIFICANCE: This study discovers that dynamic loss of terminal sialylation in glycoproteins of CTC clusters contributes to the fate of cellular dormancy, advantageous evasion to chemotherapy, and enhanced metastatic seeding. It identifies PODXL as a glycoprotein substrate of ST6GAL1 and a candidate target to counter chemoevasion-associated metastasis of quiescent tumor cells. This article is featured in Selected Articles from This Issue, p. 1949.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including convalescent COVID-19 and sero-negative controls. Flow cytometry analyses revealed reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells within the patients who have recovered from severe COVID-19. sc-RNA seq analysis identifies seven heterogeneous clusters of monocytes and nine Treg clusters featuring distinct molecular signatures in association with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocytes and Tregs expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters featuring S100 family genes: one monocyte cluster of S100A8 & A9 coupled with high HLA-I and another cluster of S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, as well as a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-lived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (≥ 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis.

Metastasis is the cause of over 90% of all deaths associated with breast cancer, yet the strategies to predict cancer spreading based on primary tumor profiles and therefore prevent metastasis are egregiously limited. As rare precursor cells to metastasis, circulating tumor cells (CTCs) in multicellular clusters in the blood are 20-50 times more likely to produce viable metastasis than single CTCs. However, the molecular mechanisms underlying various CTC clusters, such as homotypic tumor cell clusters and heterotypic tumor-immune cell clusters, are yet to be fully elucidated. Combining machine learning-assisted computational ranking with experimental demonstration to assess cell adhesion candidates, we identified a transmembrane protein Plexin- B2 (PB2) as a new therapeutic target that drives the formation of both homotypic and heterotypic CTC clusters. High PB2 expression in human primary tumors predicts an unfavorable distant metastasis-free survival and is enriched in CTC clusters compared to single CTCs in advanced breast cancers. Loss of PB2 reduces formation of homotypic tumor cell clusters as well as heterotypic tumor-myeloid cell clusters in triple-negative breast cancer. Interactions between PB2 and its ligand Sema4C on tumor cells promote homotypic cluster formation, and PB2 binding with Sema4A on myeloid cells (monocytes) drives heterotypic CTC cluster formation, suggesting that metastasizing tumor cells hijack the PB2/Sema family axis to promote lung metastasis in breast cancer. Additionally, using a global proteomic analysis, we identified novel downstream effectors of the PB2 pathway associated with cancer stemness, cell cycling, and tumor cell clustering in breast cancer. Thus, PB2 is a novel therapeutic target for preventing new metastasis.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Electroacupuncture alleviates orofacial allodynia and anxiety-like behaviors by regulating synaptic plasticity of the CA1 hippocampal region in a mouse model of trigeminal neuralgia.

Objective: Trigeminal neuralgia (TN), one of the most severe and debilitating chronic pain conditions, is often accompanied by mood disorders, such as anxiety and depression. Electroacupuncture (EA) is a characteristic therapy of Traditional Chinese Medicine with analgesic and anxiolytic effects. This study aimed to investigate whether EA ameliorates abnormal TN orofacial pain and anxiety-like behavior by altering synaptic plasticity in the hippocampus CA1. Materials and methods: A mouse infraorbital nerve transection model (pT-ION) of neuropathic pain was established, and EA or sham EA was used to treat ipsilateral acupuncture points (GV20-Baihui and ST7-Xiaguan). Golgi-Cox staining and transmission electron microscopy (TEM) were administrated to observe the changes of synaptic plasticity in the hippocampus CA1. Results: Stable and persistent orofacial allodynia and anxiety-like behaviors induced by pT-ION were related to changes in hippocampal synaptic plasticity. Golgi stainings showed a decrease in the density of dendritic spines, especially mushroom-type dendritic spines, in hippocampal CA1 neurons of pT-ION mice. TEM results showed that the density of synapses, membrane thickness of the postsynaptic density, and length of the synaptic active zone were decreased, whereas the width of the synaptic cleft was increased in pT-ION mice. EA attenuated pT-ION-induced orofacial allodynia and anxiety-like behaviors and effectively reversed the abnormal changes in dendritic spines and synapse of the hippocampal CA1 region. Conclusion: EA modulates synaptic plasticity of hippocampal CA1 neurons, thereby reducing abnormal orofacial pain and anxiety-like behavior. This provides evidence for a TN treatment strategy.

Machine learning-assisted elucidation of CD81-CD44 interactions in promoting cancer stemness and extracellular vesicle integrity.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in Convalescent COVID-19 patients.

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-ß high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of the coronavirus induced disease 2019 (COVID-19) with evolving variants of concern. It remains urgent to identify novel approaches against broad strains of SARS-CoV-2, which infect host cells via the entry receptor angiotensin-converting enzyme 2 (ACE2). Herein, we report an increase in circulating extracellular vesicles (EVs) that express ACE2 (evACE2) in plasma of COVID-19 patients, which levels are associated with severe pathogenesis. Importantly, evACE2 isolated from human plasma or cells neutralizes SARS-CoV-2 infection by competing with cellular ACE2. Compared to vesicle-free recombinant human ACE2 (rhACE2), evACE2 shows a 135-fold higher potency in blocking the binding of the viral spike protein RBD, and a 60- to 80-fold higher efficacy in preventing infections by both pseudotyped and authentic SARS-CoV-2. Consistently, evACE2 protects the hACE2 transgenic mice from SARS-CoV-2-induced lung injury and mortality. Furthermore, evACE2 inhibits the infection of SARS-CoV-2 variants (α, ß, and δ) with equal or higher potency than for the wildtype strain, supporting a broad-spectrum antiviral mechanism of evACE2 for therapeutic development to block the infection of existing and future coronaviruses that use the ACE2 receptor.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer.

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells.

Protein stability is largely regulated by post-translational modifications, such as ubiquitination, which is mediated by ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 with substrate specificity. Membrane-associated RING-CH (MARCH) proteins represent one novel family of transmembrane E3 ligases which target glycoproteins for lysosomal destruction. While most of the MARCH family members are known to degrade membrane proteins in immune cells, their tumor-intrinsic role is largely unknown. In this study, we found that the expression of one MARCH family member, MARCH8, is specifically downregulated in breast cancer tissues and positively correlated with breast cancer survival rate according to bioinformatic analysis of The Cancer Genomic Atlas (TCGA) dataset. MARCH8 protein expression was also lower in a variety of human breast cancer cell lines in comparison to immortalized human mammary epithelial MCF-12A cells. Restoration of MARCH8 expression induced apoptosis in human breast cancer cell lines MDA-MB-231 and BT549. Stable expression of MARCH8 inhibited tumorigenesis and lung metastases of MDA-MB-231 cells in mice. Moreover, we discovered that the breast cancer stem-cell marker and metastasis driver CD44, a membrane protein, interacts with MARCH8 and is one of the glycoprotein targets subject to MARCH8-dependent lysosomal degradation. Unexpectedly, we identified a nonmembrane protein, signal transducer and transcription activator 3 (STAT3), as another essential ubiquitination target of MARCH8, whose degradation through the proteasome pathway is responsible for the proapoptotic changes mediated by MARCH8. These findings highlight a novel tumor-suppressing function of MARCH8 in targeting both membrane and nonmembrane protein targets required for the survival and metastasis of breast cancer cells.

EGFR inhibition blocks cancer stem cell clustering and lung metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive and metastatic breast cancer subtypes lacking targeted therapy. Our recent work demonstrated that circulating tumor cell (CTC) clusters and polyclonal metastasis of TNBC are driven by aggregation of CD44+ cancer stem cells (CSC) and associated with an unfavorable prognosis, such as low overall survival. However, there is no existing therapeutic that can specifically block CTC or CSC cluster formation. Methods: Using patient-derived xenograft (PDX) models, we established an ex vivo tumor cell clustering assay for a pilot screening of blockade antibodies. After identifying EGFR as a target candidate, we modulated the gene expression and inhibited its kinase activity to determine its functional importance in tumor cell clustering and therapeutic inhibition of lung metastasis. We also examined the molecular regulation network of EGFR and a potential connection to CSC marker CD44 and microRNAs, which regulate CTC clustering. Results: We report here that EGFR inhibition successfully blocks circulating CSC (cCSC) clustering and lung metastasis of TNBC. EGFR enhances CD44-mediated tumor cell aggregation and CD44 stabilizes EGFR. Importantly, blocking EGFR by a novel anti-EGFR monoclonal antibody (clone LA1) effectively blocked cell aggregation in vitro and reduced lung metastasis in vivo. Furthermore, our data demonstrated that the tumor suppressor microRNA-30c serves as another negative regulator of cCSC clustering and lung metastasis by targeting CD44 as well as its downstream effector EGFR. Conclusion: Our studies identify a novel anti-EGFR therapeutic strategy to inhibit cCSC aggregation and therefore abolish cCSC cluster-mediated metastasis of TNBC.

Surfactant-assisted one-pot sample preparation for label-free single-cell proteomics.

Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-ß-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for 'all-in-one' one-pot sample preparation. This 'all-in-one' method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.

Extracellular Domains I and II of cell-surface glycoprotein CD44 mediate its trans-homophilic dimerization and tumor cluster aggregation.

CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be fully evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning-based computational modeling combined with experimental mutagenesis tests revealed that the extracellular Domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in Domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in Domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro Importantly, the substitutions in Domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer-dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

Mouse Cardiac Pde1C Is a Direct Transcriptional Target of Pparα.

Phosphodiesterase 1C (PDE1C) is expressed in mammalian heart and regulates cardiac functions by controlling levels of second messenger cyclic AMP and cyclic GMP (cAMP and cGMP, respectively). However, molecular mechanisms of cardiac Pde1c regulation are currently unknown. In this study, we demonstrate that treatment of wild type mice and H9c2 myoblasts with Wy-14,643, a potent ligand of nuclear receptor peroxisome-proliferator activated receptor alpha (PPARα), leads to elevated cardiac Pde1C mRNA and cardiac PDE1C protein, which correlate with reduced levels of cAMP. Furthermore, using mice lacking either Pparα or cardiomyocyte-specific Med1, the major subunit of Mediator complex, we show that Wy-14,643-mediated Pde1C induction fails to occur in the absence of Pparα and Med1 in the heart. Finally, using chromatin immunoprecipitation assays we demonstrate that PPARα binds to the upstream Pde1C promoter sequence on two sites, one of which is a palindrome sequence (agcTAGGttatcttaacctagc) that shows a robust binding. Based on these observations, we conclude that cardiac Pde1C is a direct transcriptional target of PPARα and that Med1 may be required for the PPARα mediated transcriptional activation of cardiac Pde1C.

PIMT/NCOA6IP Deletion in the Mouse Heart Causes Delayed Cardiomyopathy Attributable to Perturbation in Energy Metabolism.

PIMT/NCOA6IP, a transcriptional coactivator PRIP/NCOA6 binding protein, enhances nuclear receptor transcriptional activity. Germline disruption of PIMT results in early embryonic lethality due to impairment of development around blastocyst and uterine implantation stages. We now generated mice with Cre-mediated cardiac-specific deletion of PIMT (csPIMT-/-) in adult mice. These mice manifest enlargement of heart, with nearly 100% mortality by 7.5 months of age due to dilated cardiomyopathy. Significant reductions in the expression of genes (i) pertaining to mitochondrial respiratory chain complexes I to IV; (ii) calcium cycling cardiac muscle contraction (Atp2a1, Atp2a2, Ryr2); and (iii) nuclear receptor PPAR- regulated genes involved in glucose and fatty acid energy metabolism were found in csPIMT-/- mouse heart. Elevated levels of Nppa and Nppb mRNAs were noted in csPIMT-/- heart indicative of myocardial damage. These hearts revealed increased reparative fibrosis associated with enhanced expression of Tgfß2 and Ctgf. Furthermore, cardiac-specific deletion of PIMT in adult mice, using tamoxifen-inducible Cre-approach (TmcsPIMT-/-), results in the development of cardiomyopathy. Thus, cumulative evidence suggests that PIMT functions in cardiac energy metabolism by interacting with nuclear receptor coactivators and this property could be useful in the management of heart failure.