Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer.

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.

Different modes of Notch activation and strength regulation in the spermathecal secretory lineage.

The strength of Notch signaling contributes to pleiotropic actions of Notch; however, we do not yet have a full understanding of the molecular regulation of Notch-signaling strength. We have investigated the mode of Notch activation in binary fate specification in the Drosophila spermathecal linage, where Notch is asymmetrically activated across three divisions to specify different cell fates. Using clonal analysis, we show that Delta (Dl) serves as the ligand for Notch in the first and second divisions. Dl and Serrate (Ser) function redundantly in the third division. Compared with the third division, cell-fate decision in the second division requires a lower level of Suppressor of Hairless protein, and, consequently, a lower level of Notch signaling. Several Notch endosomal trafficking regulators differentially regulate Notch signaling between the second and third divisions. Here, we demonstrate that cell differentiation in spermathecae involves different Notch-activation modes, Notch-signaling strengths and Notch-trafficking regulations. Thus, the Drosophila spermathecal lineage is an exciting model for probing the molecular mechanisms that modulate the Notch signaling pathway.

A splicing factor switch controls hematopoietic lineage specification of pluripotent stem cells.

Alternative splicing (AS) leads to transcriptome diversity in eukaryotic cells and is one of the key regulators driving cellular differentiation. Although AS is of crucial importance for normal hematopoiesis and hematopoietic malignancies, its role in early hematopoietic development is still largely unknown. Here, by using high-throughput transcriptomic analyses, we show that pervasive and dynamic AS takes place during hematopoietic development of human pluripotent stem cells (hPSCs). We identify a splicing factor switch that occurs during the differentiation of mesodermal cells to endothelial progenitor cells (EPCs). Perturbation of this switch selectively impairs the emergence of EPCs and hemogenic endothelial progenitor cells (HEPs). Mechanistically, an EPC-induced alternative spliced isoform of NUMB dictates EPC specification by controlling NOTCH signaling. Furthermore, we demonstrate that the splicing factor SRSF2 regulates splicing of the EPC-induced NUMB isoform, and the SRSF2-NUMB-NOTCH splicing axis regulates EPC generation. The identification of this splicing factor switch provides a new molecular mechanism to control cell fate and lineage specification.

Numb inhibits migration and promotes proliferation of colon cancer cells via RhoA/ROCK signaling pathway repression.

Numb regulates cell proliferation and differentiation through endocytosis and ubiquitination of signaling molecules. Besides, Numb controls the migration of epithelial cells by regulating intercellular junctions. Studies have shown that Numb promotes or inhibits tumor progression in different tumors. However, its role and mechanism in colorectal cancer remain unclear. We found that the expression level of Numb in colon tumor tissues has a great variety in different patients. Numb expression was negatively correlated with TNM stage and lymph node metastasis but positively correlated with tumor size. Elevated expression of Numb was associated with a good prognosis. Inhibiting Numb expression promoted the migration and invasion of colon cancer cells induced by TGF-ß, up-regulated the expression of EMT-related molecule Snail, and prevented the expression of E-cadherin. We also found that Numb promoted the proliferation and clones formation while inhibiting colon cancer cells' late apoptosis. In addition, Numb inhibited the RhoA activation and ROCK inhibitor Y-27632 or interfered with ROCK expression, partially inhibiting Numb-regulated cell proliferation and migration. In vivo tumorigenesis assay in nude mice also found that Numb promoted the proliferation of colon cancer cells, inhibited the expression of E-cadherin, and strengthened the expression of Snail. In conclusion, our study found that Numb plays multiple roles in the occurrence and progression of colon cancer by regulating the RhoA/ROCK signaling pathway, which provides a new theoretical molecular basis for the pathogenesis of colon cancer.

Denervation Atrophy of Skeletal Muscle is Not Influenced by Numb Levels in Mice.

Skeletal muscle undergoes rapid and extensive atrophy following nerve transection though the underlying mechanisms remain incompletely understood. We previously showed transiently elevated Notch 1 signaling in denervated skeletal muscle that was abrogated by administration of nandrolone (an anabolic steroid) combined with replacement doses of testosterone. Numb is an adaptor molecule present in myogenic precursors and skeletal muscle fibers that is vital for normal tissue repair after muscle injury and for skeletal muscle contractile function. It is unclear whether the increase in Notch signaling observed in denervated muscle contributes to denervation and whether expression of Numb in myofibers slows denervation atrophy. To address these questions, the degree of denervation atrophy, Notch signaling, and Numb expression was studied over time after denervation in C57B6J mice treated with nandrolone, nandrolone plus testosterone or vehicle. Nandrolone increased Numb expression and reduced Notch signaling. Neither nandrolone alone nor nandrolone plus testosterone changed the rate of denervation atrophy. We next compared rates of denervation atrophy between mice with conditional, tamoxifen-inducible knockout of Numb in myofibers and genetically identical mice treated with vehicle. Numb cKO had no effect on denervation atrophy in this model. Taken together, the data indicate that loss of Numb in myofibers does not alter the course of denervation atrophy and that upregulation of Numb and blunting of the denervation-atrophy induced activation of Notch do not change the course of denervation atrophy.

Study on lactation performance and development of KASP marker for milk traits in Xinjiang donkey (Equus asinus).

Donkey milk has high nutritional and medicinal value, but there are few researches in donkey milk traits, especially on genome. The whole lactation of 89 donkeys was recorded and it was found that Xinjiang donkey had good lactation performance while great differences among individuals. In our previous study, four genes including LGALS2, NUMB, ADCY8 and CA8 were identified as milk-associated with Chinese Kazakh house, based on Equine 670k Chip genomic analysis. And then 15 SNPs of the four key genes were conducted for genotyping in Xinjiang donkey in this study, one of Chinese indigenous breed, 14 SNPs were successful classified. And those SNPs were correlation analysis with milk yield of Xinjiang donkeys. The results showed that NUMB g.46709914T > G was significantly correlated with daily milk yield of Xinjiang donkey in the early, middle, and late periods, while ADCY8 g.48366302T > C, CA8 g.89567442T > G and CA8 g.89598328T > A were significantly correlated with lactation in the late periods. These results indicate that NUMB g.46709914T > G can be as markers of candidate genes for lactating traits in donkeys, SNPs of ADCY8 and CA8 as potential. Our findings will not only help confirm key genes for donkey milk traits, but also provide future for genomic selection in donkeys.

Multifunctional protein 4.1R regulates the asymmetric segregation of Numb during terminal erythroid maturation.

The asymmetric cell division of stem or progenitor cells generates daughter cells with distinct fates that balance proliferation and differentiation. Asymmetric segregation of Notch signaling regulatory protein Numb plays a crucial role in cell diversification. However, the molecular mechanism remains unclear. Here, we examined the unequal distribution of Numb in the daughter cells of murine erythroleukemia cells (MELCs) that undergo DMSO-induced erythroid differentiation. In contrast to the cytoplasmic localization of Numb during uninduced cell division, Numb is concentrated at the cell boundary in interphase, near the one-spindle pole in metaphase, and is unequally distributed to one daughter cell in anaphase in induced cells. The inheritance of Numb guides this daughter cell toward erythroid differentiation while the other cell remains a progenitor cell. Mitotic spindle orientation, critical for distribution of cell fate determinants, requires complex communication between the spindle microtubules and the cell cortex mediated by the NuMA-LGN-dynein/dynactin complex. Depletion of each individual member of the complex randomizes the position of Numb relative to the mitotic spindle. Gene replacement confirms that multifunctional erythrocyte protein 4.1R (4.1R) functions as a member of the NuMA-LGN-dynein/dynactin complex and is necessary for regulating spindle orientation, in which interaction between 4.1R and NuMA plays an important role. These results suggest that mispositioning of Numb is the result of spindle misorientation. Finally, disruption of the 4.1R-NuMA-LGN complex increases Notch signaling and decreases the erythroblast population. Together, our results identify a critical role for 4.1R in regulating the asymmetric segregation of Numb to mediate erythropoiesis.

Numb deficiency impairs retinal structure and visual function in mice.

PURPOSE: Numb is expressed in the progenitor and mature neurons throughout the development progress of the retina. We attempted to investigate the role of Numb in the retina and visual system, and the effects of Numb deficiency on retinal structure and visual function. METHODS: Conditional Numb/Nbl double-knockout mice were generated to observe the retinal damage in Numb deficiency mice. HE staining and immunofluorescence stain were used to observe the structural and molecular changes. The visual function was assessed by electroretinogram (ERG) and optomotor response (OMR). RNA-Seq and RT-PCR were used to detect the differential expression of genes and the related signaling pathways. RESULTS: Inactivation of Numb/Nbl induces eye apoptosis and retinal neurons impairment observed by HE staining and immunofluorescence stain. The impaired retinal structure and visual function were assessed by ERG and OMR. RNA-seq analysis indicated loss of photoreceptors, synapses and phototransduction related molecules. Immunofluorescence stain of molecular markers recoverin, arrestin, rhodopsin showed disrupted structural integrity of photoreceptors. Additional bipolar cells and synapses related molecular markers indicate synaptic connections were damaged in Numb deficiency mice. CONCLUSIONS: Inactivation of Numb/Nbl induces eye apoptosis and retinal neurons impairment. Ablation of Numb in retina significantly impaired visual function. The impaired visual function in Numb deficiency mice is related to the damage of photoreceptors, ion/cation channel activity, synapse formation and phototransduction.

Cardiomyocyte orientation modulated by the Numb family proteins-N-cadherin axis is essential for ventricular wall morphogenesis.

The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.

Melatonin Inhibits 17ß-Estradiol-Induced Epithelial-Mesenchymal Transition in Endometrial Adenocarcinoma Cells via Upregulating Numb Expression.

OBJECTIVES: Melatonin (MLT) shows antitumor effects in various tumor types, including endometrial carcinoma. However, the molecular mechanism involved is unclear. In the current study, we investigated the effect of MLT on the estrogen-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells and explored the pathway that might be involved. DESIGN: Laboratory study was via cultured endometrial cancer cells. Design refers only to in vitro experiments. METHODS: In cell culture experiments, cell growth was examined using CCK-8 assays. The expression of Numb and EMT markers in Ishikawa cells was examined using Western blot analysis and real-time PCR. Cell invasion was examined using transwell assays. Cell migration was examined using wound-healing assays and transwell assays. Using immunohistochemistry analysis, the expression of Numb in human endometrial cancers was examined. RESULTS: In immunohistochemistry experiments, we found that 15.2% of atypical endometrial hyperplasia and 15.6% of endometrial carcinoma did not express Numb. In cell culture experiments, MLT inhibited cell proliferation, invasion, and migration induced by 17ß-estradiol (E2) in endometrial cancer cells. MLT decreased the expression of vimentin and Slug and increased the expression of Numb and E-cadherin in Ishikawa cells. Numb knockdown in cancer cells significantly increased cell proliferation, invasion, and migration. LIMITATIONS: No animal experiments were performed. CONCLUSIONS: MLT blocked E2-induced cell growth and EMT in endometrial cancer cells via upregulating Numb expression.

Exclusion of NUMB Exon12 Controls Cancer Cell Migration through Regulation of Notch1-SMAD3 Crosstalk.

NUMB is an endocytic adaptor protein that contains four isoforms (p65, p66, p71 and p72) due to alternative splicing regulation. Here, we show that NUMB exon12 (E12)-skipping isoforms p65/p66 promote epithelial to mesenchymal transition (EMT) and cancer cell migration in vitro, and facilitate cancer metastasis in mice, whereas E12-included p71/p72 isoforms attenuate these effects. Mechanistically, p65/p66 isoforms significantly increase the sorting of Notch1 through early endosomes (EEs) for enhanced Notch1 activity. In contrast, p71/p72 isoforms act as negative regulators of Notch1 by ubiquitylating the Notch1 intracellular domain (N1ICD) and promoting its degradation. Moreover, we observed that the interaction between N1ICD and SMAD3 is important for their own stabilization, and for NUMB-mediated EMT response and cell migration. Either N1ICD or SMAD3 overexpression could significantly recuse the migration reduction seen in the p65/p66 knockdown, and Notch1 or SMAD3 knockdown rescued the migration advantage seen in the overexpression of p66. Taken all together, our study provides mechanistic insights into the opposite regulation of Notch1-SMAD3 crosstalk by NUMB isoforms and identifies them as critical regulators of EMT and cancer cell migration.

Intrinsic regulation of enteroendocrine fate by Numb.

How terminal cell fates are specified in dynamically renewing adult tissues is not well understood. Here we explore terminal cell fate establishment during homeostasis using the enteroendocrine cells (EEs) of the adult Drosophila midgut as a paradigm. Our data argue against the existence of local feedback signals, and we identify Numb as an intrinsic regulator of EE fate. Our data further indicate that Numb, with alpha-adaptin, acts upstream or in parallel of known regulators of EE fate to limit Notch signaling, thereby facilitating EE fate acquisition. We find that Numb is regulated in part through its asymmetric and symmetric distribution during stem cell divisions; however, its de novo synthesis is also required during the differentiation of the EE cell. Thus, this work identifies Numb as a crucial factor for cell fate choice in the adult Drosophila intestine. Furthermore, our findings demonstrate that cell-intrinsic control mechanisms of terminal cell fate acquisition can result in a balanced tissue-wide production of terminally differentiated cell types.

Numb chin syndrome in sickle cell disease: a case series of Jamaican patients.

Numb chin syndrome is an uncommon presentation that has been reported as secondary to metastatic disease, trauma, and infections of the maxilla, mandible, or oral cavity. The hypoesthesia, paraesthesia, or pain are a result of injury to the inferior alveolar nerve, which is particularly vulnerable as it exits the mandible through the mandibular foramen as the mental nerve. In persons with sickle cell disease, it has been reported as a manifestation of mandibular vaso-occlusive crisis. This case series presents 13 patients with sickle cell disease who presented with numb chin syndrome, the largest number of cases that has been described in the literature to date. The report illustrates the wide variety of presentations and therefore possible differential diagnoses to consider. In this case series, the symptoms were associated with vaso-occlusive crises, allergic reactions, dental infections, malignancy, rheumatoid arthritis, and pregnancy. Most appeared to be self-limiting; however, one patient was having his second episode, and the numbness has persisted in three patients. The series illustrates that it is important not only to ensure that the source of the local vaso-occlusive crisis is treated, but also to not miss important differentials such as metastatic disease, where this can be the first presentation of malignancy and would represent a very poor prognosis. There is no reported successful treatment for the hypoesthesia in this case series, and this presents an area for further research.

Different roles of Numb-p72 and Numb-p65 on the trafficking of metabotropic glutamate receptor 5.

We previously reported that Numb, a protein localized to clathrin-coated vesicles, regulates the membrane expression of metabotropic glutamate receptor 5 (mGluR5) and is critical to social behaviors. However, the distinct actions of Numb isoforms on mGluR5 have not been investigated. Here, we showed that the expression patterns of Numb-p72 and Numb-p65, two important isoforms of Numb, were distinct in HEK293T cells. Numb-p72, but not Numb-p65, bound to mGluR5α, and enhanced mGluR5 membrane expression by inhibiting its internalization. Our results suggest that a complete structure is required for Numb to bind to mGluR5 and to modulate mGluR5 trafficking.

Numb chin syndrome as a presentation for acute myeloid leukemia relapse post-hematopoietic cell transplant: Case report and review of literature.

NCS is defined as reduced or absent sensation in the chin and lower lip within the distribution of the mental or inferior alveolar nerves. Although commonly associated with local trauma, NCS can indicate an underlying malignancy or can be the presenting symptom of cancer recurrence. We describe a 6-year-old female patient with AML and t(8,21) who underwent allogeneic HCT. Five months post-HCT, the patient presented with right-sided facial pain and numbness in her chin and lower lip consistent with NCS. Two weeks later, the patient had bone marrow relapse indicating AML recurrence. Although NCS remains a rare diagnosis especially in children, in the context of leukemia, it usually indicates an advanced disease or could be a sign of recurrence, and is commonly associated with grim prognosis. Further research is needed to study the link between NCS and specific cytogenetic abnormalities.

miR-31-NUMB Cascade Modulates Monocarboxylate Transporters to Increase Oncogenicity and Lactate Production of Oral Carcinoma Cells.

Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated death worldwide. miR-31 is an oncogenic miRNA in OSCC. NUMB is an adaptor protein capable of suppressing malignant transformation. Disruption of the miR-31-NUMB regulatory axis has been demonstrated in malignancies. Mitochondrial dysfunction and adaptation to glycolytic respiration are frequent events in malignancies. Monocarboxylate transporters (MCTs) function to facilitate lactate flux in highly glycolytic cells. Upregulation of MCT1 and MCT4 has been shown to be a prognostic factor of OSCC. Here, we reported that miR-31-NUMB can modulate glycolysis in OSCC. Using the CRISPR/Cas9 gene editing strategy, we identified increases in oncogenic phenotypes, MCT1 and MCT4 expression, lactate production, and glycolytic respiration in NUMB-deleted OSCC subclones. Transfection of the Numb1 or Numb4 isoform reversed the oncogenic induction elicited by NUMB deletion. This study also showed, for the first time, that NUMB4 binds MCT1 and MCT4 and that this binding increases their ubiquitination, which may decrease their abundance in cell lysates. The disruptions in oncogenicity and metabolism associated with miR-31 deletion and NUMB deletion were partially rescued by MCT1/MCT4 expression or knockdown. This study demonstrated that NUMB is a novel binding partner of MCT1 and MCT4 and that the miR-31-NUMB-MCT1/MCT4 regulatory cascade is present in oral carcinoma.

Complex wall materials of polysaccharide and protein effectively protected numb-taste substance degradation of Zanthoxylum bungeanum.

BACKGROUND: Hydroxyl-sanshools are mainly responsible for the numb taste and biological activities of Zanthoxylum bungeanum, but they show low water solubility, high volatility and easy degradation, which limit their application in the catering and food industries. Thus microcapsules of Z. bungeanum essential oil (ZBEO) were prepared to prevent numb-taste substance attenuation. RESULTS: The complex effects of hydroxypropyl-ß-cyclodextrin (HPCD) with other materials, such as konjac glucomannan octenyl succinate (KGOS), octenyl succinic anhydride-modified starch (OSAS), soy protein isolate (SPI) and gum arabic (GA), on the protection of the main numb-taste substance of ZBEO were investigated. Scanning electron microscopy and Fourier transform infrared spectroscopy analysis indicated that ZBEO was successfully encapsulated in the complex wall materials. X-ray diffraction indicated that the loaded essential oil did not affect the crystalline form of the wall material. The stability of the numb-taste substance α-sanshool in the microcapsules prepared with the complex microcapsule wall materials was higher than that in single-wall microcapsules. Storage stability evaluation indicated that microcapsules prepared with a combination of HPCD and SPI showed the greatest effect in maintaining the stability of the main numb-taste substance α-sanshool in ZBEO at room temperature, low pH and in high-salt conditions. CONCLUSION: Complex wall materials of polysaccharide and protein could effectively protect the numb-taste substance degradation of Z. bungeanum during processing and storage. © 2021 Society of Chemical Industry.

Is the unstable ataxic hand of Alajouanine and Akerman a distinct contribution?

We discuss from a historical perspective whether the 1931 description of the "unstable ataxic hand" by Théophile Alajouanine, the fifth successor of Charcot at la Salpêtrière, and the Brazilian neurologist Abraham Akerman, then studying in France, merits being considered a distinct contribution vis-à-vis the earlier description by Oppenheim of the "useless hand syndrome". The specific object of the article by Alajouanine and Akerman was a semiologic sign, namely a pseudoathetosis localized in the hand, while the original description by Oppenheim of the symptom-complex that came to be known as useless hand syndrome did not include an abnormal movement. Moreover, as a result of the useless hand syndrome originating from a clinical classification of multiple sclerosis based on the localization of the lesions, it involves topographic and etiologic diagnoses specificities. In contrast, the unstable ataxic hand can be observed in the useless hand syndrome and other syndromes involving predominantly sensory symptoms, such as "numb clumsy hands" due to high cervical spondylosis or extramedullary tumor, and the "cortical sensory syndrome" most commonly due to parietal stroke. Because it had not been thoroughly described in the context of a symptom-complex, Alajouanine and Akerman's unstable ataxic hand merits being considered a distinct and valuable contribution.

Metastatic prostate cancer presenting as trigeminal nerve paresthesia in the mandible.

Metastatic disease to the oral cavity is rare and often presents with ambiguous symptoms and subtle or no radiographic changes. These factors make diagnosis challenging. This article describes a case of metastatic prostate cancer to the mandible that presented as altered sensation to the gingiva, lips, and chin without radiographic evidence of pathosis noted on standard dental imaging. At the time of presentation, the patient's prostate cancer, diagnosed 9 years previously, was thought to be well-controlled with medical therapy. With additional laboratory testing and medical imaging, widespread metastatic disease was discovered. This case reinforces the importance of including metastatic disease in the differential diagnosis as well as the utility of collaboration with providers outside dentistry.

Eps15R and clathrin regulate EphB2-mediated cell repulsion.

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans-endocytosis of the inter-cellular receptor-ligand EphB-ephrinB complex is required. The molecular mechanism underlying trans-endocytosis is poorly defined. Here we show that the process is clathrin- and Eps15R-mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co-cultures of EphB2- and ephrinB1-expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2-mediated cell repulsion as shown in a rescue experiment in the EphB2 co-culture assay where wild type Eps15R but not the clathrin-binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans-endocytosis and thereby cell repulsion.