Severe Combined Immunodeficiency from a Homozygous DNA Ligase 1 Mutant with Reduced Catalytic Activity but Increased Ligation Fidelity.

A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

PD-L1 intrinsically promotes the proliferation of breast cancer cells through the SKP2-p27/p21 axis.

BACKGROUND: PD-L1 intrinsically promotes tumor progression through multiple mechanisms, which potentially leads to resistance to anti-PD-1/PD-L1 therapies. The intrinsic effect of PD-L1 on breast cancer (BC) cell proliferation has not been fully elucidated. METHODS: we used proteomics, gene expression knockdown (KD), quantitative immunofluorescence (qIF), western blots, functional assays including colony-forming assay (CFA) and real-time cell analyzer (RTCA), and in vivo data using immunohistochemistry in breast cancer patients. RESULTS: PD-L1 promoted BC cell proliferation by accelerating cell cycle entry at the G1-to-S phase transition. Global proteomic analysis of the differentially expressed nuclear proteins indicated the involvement of several proliferation-related molecules, including p21CIP1/WAF1. Western blotting and qIF demonstrated the higher expression of SKP2 and the lower expression of p21CIP1/WAF1 and p27Kip1 in PD-L1 expressing (PD-L1pos) cells as compared to PD-L1 KD (PD-L1KD) cells. Xenograft-derived cells and the TCGA BC dataset confirmed this relationship in vivo. Functionally, CFA and RTCA demonstrated the central role of SKP2 in promoting PD-L1-mediated proliferation. Finally, immunohistochemistry in 74 breast cancer patients confirmed PD-L1 and SKP-p21/p27 axis relationship, as it showed a highly statistically significant correlation between SKP2 and PD-L1 expression (p < 0.001), and both correlated significantly with the proliferation marker Ki-67 (p < 0.001). On the other hand, there was a statistically significant inverse relationship between PD-L1 and p21CIP1/WAF1 expression (p = 0.005). Importantly, double negativity for p21CIP1/WAF1 and p27Kip1 correlated significantly with PD-L1 (p < 0.001), SKP2 (p = 0.002), and Ki-67 (p = 0.002). CONCLUSIONS: we have demonstrated the role of the SKP2-p27/p21 axis in intrinsic PD-L1-enhanced cell cycle progression. Inhibitors of SKP2 expression can alleviate resistance to ICPIs.

Reciprocal interplays between MicroRNAs and pluripotency transcription factors in dictating stemness features in human cancers.

The interplay between microRNAs (miRNAs) and pluripotency transcription factors (TFs) orchestrates the acquisition of cancer stem cell (CSC) features during the course of malignant transformation, rendering them essential cancer cell dependencies and therapeutic vulnerabilities. In this review, we discuss emerging themes in tumor heterogeneity, including the clonal evolution and the CSC models and their implications in resistance to cancer therapies, and then provide thorough coverage on the roles played by key TFs in maintaining normal and malignant stem cell pluripotency and plasticity. In addition, we discuss the reciprocal interactions between miRNAs and MYC, OCT4, NANOG, SOX2, and KLF4 pluripotency TFs and their contributions to tumorigenesis. We provide our view on the potential to interfere with key miRNA-TF networks through the use of RNA-based therapeutics as single agents or in combination with other therapeutic strategies, to abrogate the CSC state and render tumor cells more responsive to standard and targeted therapies.

Fascin is essential for mammary gland lactogenesis.

Fascin expression has commonly been observed in certain subtypes of breast cancer, where its expression is associated with poor clinical outcome. However, its role in normal mammary gland development has not been elucidated. Here, we used a fascin knockout mouse model to assess its role in normal mammary gland morphogenesis and lactation. Fascin knockout was not embryonically lethal, and its effect on the litter size or condition at birth was minimal. However, litter survival until the weaning stage significantly depended on fascin expression solely in the nursing dams. Accordingly, pups that nursed from fascin-/- dams had smaller milk spots in their abdomen, suggesting a lactation defect in the nursing dams. Mammary gland whole-mounts of pregnant and lactating fascin-/- mice showed significantly reduced side branching and alveologenesis. Despite a typical composition of basal, luminal, and stromal subsets of mammary cells and normal ductal architecture of myoepithelial and luminal layers, the percentage of alveolar progenitors (ALDH+) in fascin-/- epithelial fraction was significantly reduced. Further in-depth analyses of fascin-/- mammary glands showed a significant reduction in the expression of Elf5, the master regulator of alveologenesis, and a decrease in the activity of its downstream target p-STAT5. In agreement, there was a significant reduction in the expression of the milk proteins, whey acidic protein (WAP), and ß-casein in fascin-/- mammary glands. Collectively, our data demonstrate, for the first time, the physiological role of fascin in normal mammary gland lactogenesis, an addition that could reveal its contribution to breast cancer initiation and progression.

E. coli Secretome Metabolically Modulates MDA-MB-231 Breast Cancer Cells' Energy Metabolism.

Breast cancer (BC) is commonly diagnosed in women. BC cells are associated with altered metabolism, which is essential to support their energetic requirements, cellular proliferation, and continuous survival. The altered metabolism of BC cells is a result of the genetic abnormalities of BC cells. Risk factors can also enhance it, including age, lifestyle, hormone disturbances, etc. Other unknown BC-promoting risk factors are under scientific investigation. One of these investigated factors is the microbiome. However, whether the breast microbiome found in the BC tissue microenvironment can impact BC cells has not been studied. We hypothesized that E. coli, part of a normal breast microbiome with more presence in BC tissue, secretes metabolic molecules that could alter BC cells' metabolism to maintain their survival. Thus, we directly examined the impact of the E. coli secretome on the metabolism of BC cells in vitro. MDA-MB-231 cells, an in vitro model of aggressive triple-negative BC cells, were treated with the E. coli secretome at different time points, followed by untargeted metabolomics analyses via liquid chromatography-mass spectrometry to identify metabolic alterations in the treated BC cell lines. MDA-MB-231 cells that were not treated were used as controls. Moreover, metabolomic analyses were performed on the E. coli secretome to profile the most significant bacterial metabolites affecting the metabolism of the treated BC cell lines. The metabolomics results revealed about 15 metabolites that potentially have indirect roles in cancer metabolism that were secreted from E. coli in the culture media of MDA-MB-231 cells. The cells treated with the E. coli secretome showed 105 dysregulated cellular metabolites compared to controls. The dysregulated cellular metabolites were involved in the metabolism of fructose and mannose, sphingolipids, amino acids, fatty acids, amino sugar, nucleotide sugar, and pyrimidine, which are vital pathways required for the pathogenesis of BC. Our findings are the first to show that the E. coli secretome modulates the BC cells' energy metabolism, highlighting insights into the possibility of altered metabolic events in BC tissue in the actual BC tissue microenvironment that are potentially induced by the local bacteria. Our study provides metabolic data that could be as a basis for future studies searching for the underlying mechanisms mediated by bacteria and their secretome to alter the metabolism of BC cells.

ß1 Integrin is essential for fascin-mediated breast cancer stem cell function and disease progression.

Breast cancer remains the second cause of tumor-related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem-like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin-bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, ß1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin-mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self-renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co-expression and short disease-free as well as overall survival in chemo-treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self-renewability and chemoresistance strongly encourages for dual targeting of fascin-ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root.

PD-L1 promotes OCT4 and Nanog expression in breast cancer stem cells by sustaining PI3K/AKT pathway activation.

The expression of PD-L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial-to-mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD-L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p < 0.0001, r = 0.36) between PD-L1 expression and stemness score of breast cancer. Specific knockdown of PD-L1 using ShRNA revealed its critical role in the expression of the embryonic stem cell transcriptional factors: OCT-4A, Nanog and the stemness factor, BMI1. Conversely, these factors could be induced upon PD-L1 ectopic expression in cells that are normally PD-L1 negative. Global proteomic analysis hinted for the central role of AKT in the biology of PD-L1 expressing cells. Indeed, PD-L1 positive effect on OCT-4A and Nanog was dependent on AKT activation. Most importantly, downregulation of PD-L1 compromised the self-renewal capability of breast CSCs in vitro and in vivo as shown by tumorsphere formation assay and extreme limiting dilution assay, respectively. This study demonstrates a novel role for PD-L1 in sustaining stemness of breast cancer cells and identifies the subpopulation and its associated molecular pathways that would be targeted upon anti-PD-L1 therapy.

Fascin Is Critical for the Maintenance of Breast Cancer Stem Cell Pool Predominantly via the Activation of the Notch Self-Renewal Pathway.

An emerging dogma shows that tumors are initiated and maintained by a subpopulation of cancer cells that hijack some stem cell features and thus referred to as "cancer stem cells" (CSCs). The exact mechanism that regulates the maintenance of CSC pool remains largely unknown. Fascin is an actin-bundling protein that we have previously demonstrated to be a major regulator of breast cancer chemoresistance and metastasis, two cardinal features of CSCs. Here, we manipulated fascin expression in breast cancer cell lines and used several in vitro and in vivo approaches to examine the relationship between fascin expression and breast CSCs. Fascin knockdown significantly reduced stem cell-like phenotype (CD44hi /CD24lo and ALDH+ ) and reversal of epithelial to mesenchymal transition. Interestingly, expression of the embryonic stem cell transcriptional factors (Oct4, Nanog, Sox2, and Klf4) was significantly reduced when fascin expression was down-regulated. Functionally, fascin-knockdown cells were less competent in forming colonies and tumorspheres, consistent with lower basal self-renewal activity and higher susceptibility to chemotherapy. Fascin effect on CSC chemoresistance and self-renewability was associated with Notch signaling. Activation of Notch induced the relevant downstream targets predominantly in the fascin-positive cells. Limiting-dilution xenotransplantation assay showed higher frequency of tumor-initiating cells in the fascin-positive group. Collectively, our data demonstrated fascin as a critical regulator of breast CSC pool at least partially via activation of the Notch self-renewal signaling pathway and modification of the expression embryonic transcriptional factors. Targeting fascin may halt CSCs and thus presents a novel therapeutic approach for effective treatment of breast cancer. Stem Cells 2016;34:2799-2813 Video Highlight: https://youtu.be/GxS4fJ_Ow-o.

Bidirectional crosstalk between PD-L1 expression and epithelial to mesenchymal transition: significance in claudin-low breast cancer cells.

BACKGROUND: The T-cell inhibitory molecule PD-L1 (B7-H1, CD274) is expressed on tumor cells of a subset of breast cancer patients. However, the mechanism that regulates PD-L1 expression in this group of patients is still not well-identified. METHODS: We have used loss and gain of function gene manipulation approach, multi-parametric flow cytometry, large scale gene expression dataset analysis and immunohistochemistry of breast cancer tissue sections. RESULTS: Induction of epithelial to mesenchymal transition (EMT) in human mammary epithelial cells upregulated PD-L1 expression, which was dependent mainly on the activation of the PI3K/AKT pathway. Interestingly, gene expression signatures available from large cohort of breast tumors showed a significant correlation between EMT score and the PD-L1 mRNA level (p < 0.001). Strikingly, very strong association (p < 0.0001) was found between PD-L1 expression and claudin-low subset of breast cancer, which is known to have high EMT score. On the protein level, significant correlation was found between PD-L1 expression and standard markers of EMT (p = 0.005) in 67 breast cancer patients. Importantly, specific downregulation of PD-L1 in claudin-low breast cancer cells showed signs of EMT reversal as manifested by CD44 and Vimentin downregulation and CD24 upregulation. CONCLUSIONS: We have demonstrated a bidirectional effect between EMT status and PD-L1 expression especially in claudin-low subtype of breast cancer cells. Our findings highlights the potential dual benefit of anti-PD-L1 particularly in this subset of breast cancer patients that will likely benefit more from anti-PD-L1 targeted therapy as well as in monitoring biological changes upon treatment.

A unique STK4 mutation truncating only the C-terminal SARAH domain results in a mild clinical phenotype despite severe T cell lymphopenia: Case report.

Mutations in STK4 (MST1) are implicated in a form of autosomal recessive combined immunodeficiency, resulting in recurrent infections (especially Epstein-Barr virus viremia), autoimmunity, and cardiac malformations. Here we report a patient with an atypically mild presentation of this disease, initially presenting with severe T cell lymphopenia (< 500 per mm3) and intermittent neutropenia, but now surviving well on immunoglobulins and prophylactic antibacterial treatment. She harbors a unique STK4 mutation that lies further downstream than all others reported to date. Unlike other published cases, her mRNA transcript is not vulnerable to nonsense mediated decay (NMD) and yields a truncated protein that is expected to lose only the C-terminal SARAH domain. This domain is critical for autodimerization and autophosphorylation. While exhibiting significant differences from controls, this patient's T cell proliferation defects and susceptibility to apoptosis are not as severe as reported elsewhere. Expression of PD-1 is in line with healthy controls. Similarly, the dysregulation seen in immunophenotyping is not as pronounced as in other published cases. The nature of this mutation, enabling its evasion from NMD, provides a rare glimpse into the clinical and cellular features associated with the absence of a "null" phenotype of this protein.

Differential marker expression by cultures rich in mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells have properties that make them amenable to therapeutic use. However, the acceptance of mesenchymal stem cells in clinical practice requires standardized techniques for their specific isolation. To date, there are no conclusive marker (s) for the exclusive isolation of mesenchymal stem cells. Our aim was to identify markers differentially expressed between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. We compared and contrasted the phenotype of tissue cultures in which mesenchymal stem cells are rich and rare. By initially assessing mesenchymal stem cell differentiation, we established that bone marrow and breast adipose cultures are rich in mesenchymal stem cells while, in our hands, foreskin fibroblast and olfactory tissue cultures contain rare mesenchymal stem cells. In particular, olfactory tissue cells represent non-stem cell mesenchymal cells. Subsequently, the phenotype of the tissue cultures were thoroughly assessed using immuno-fluorescence, flow-cytometry, proteomics, antibody arrays and qPCR. RESULTS: Our analysis revealed that all tissue cultures, regardless of differentiation potential, demonstrated remarkably similar phenotypes. Importantly, it was also observed that common mesenchymal stem cell markers, and fibroblast-associated markers, do not discriminate between mesenchymal stem cell and non-stem cell mesenchymal cell cultures. Examination and comparison of the phenotypes of mesenchymal stem cell and non-stem cell mesenchymal cell cultures revealed three differentially expressed markers - CD24, CD108 and CD40. CONCLUSION: We indicate the importance of establishing differential marker expression between mesenchymal stem cells and non-stem cell mesenchymal cells in order to determine stem cell specific markers.

Phosphoinositide 3-kinase-regulated adapters in lymphocyte activation.

Signaling via phosphoinositide 3-kinases (PI3Ks) has emerged as a central component of lymphocyte activation via immunoreceptors, costimulatory receptors, cytokine receptors, and chemokine receptors. The discovery of phosphoinositide-binding pleckstrin homology (PH) domains has substantially increased understanding of how PI3Ks activate cellular responses. Accumulating evidence indicates that PH-domain containing adapter molecules provide important links between PI3K and lymphocyte function. Here, we review data on PI3K-regulated adapter proteins of the Grb-associated binder (GAB), Src kinase-associated phosphoprotein (SKAP), and B-lymphocyte adapter molecule of 32 kDa (Bam32)/ dual-adapter for phosphotyrosine and 3-phosphoinositides (DAPP)/TAPP families, with a focus on the latter group. Current data support the model that recruitment of these adapters to the plasma membrane of activated lymphocytes is driven by the phosphoinositides phosphatidylinositol-3,4,5-tris-phosphate and phosphatidylinositol-3,4-bisphosphate, generated through the action of PI3Ks and under the regulatory control of lipid phosphatases Src homology 2 domain-containing inositol phosphatase (SHIP), phosphatase and tensin homolog, and inositol polyphosphate 4-phosphatase. At the plasma membrane, these adapters serve to assemble distinct protein complexes. Bam32/DAPP1 and SKAPs function to promote activation of monomeric guanosine triphosphatases, including Rac and Rap, and promote integrin activation, lymphocyte adhesion to matrix proteins, and cell:cell interactions between B and T lymphocytes. GABs can provide feedforward amplification or feedback inhibition of PI3K signaling. Current work is further defining the molecular interactions driven by these molecules and identifying the functions of TAPP adapters, which also appear to be involved in lymphocyte adhesion and are specific effectors downstream of the SHIP product phosphatidylinositol-3,4-bisphosphate.

Metabolic Alteration of MCF-7 Cells upon Indirect Exposure to E. coli Secretome: A Model of Studying the Microbiota Effect on Human Breast Tissue.

According to studies, the microbiome may contribute to the emergence and spread of breast cancer. E. coli is one of the Enterobacteriaceae family recently found to be present as part of the breast tissue microbiota. In this study, we focused on the effect of E. coli secretome free of cells on MCF-7 metabolism. Liquid chromatography-mass spectrometry (LC-MS) metabolomics was used to study the E. coli secretome and its role in MCF-7 intra- and extracellular metabolites. A comparison was made between secretome-exposed cells and unexposed controls. Our analysis revealed significant alterations in 31 intracellular and 55 extracellular metabolites following secretome exposure. Several metabolic pathways, including lactate, aminoacyl-tRNA biosynthesis, purine metabolism, and energy metabolism, were found to be dysregulated upon E. coli secretome exposure. E. coli can alter the breast cancer cells' metabolism through its secretome which disrupts key metabolic pathways of MCF-7 cells. These microbial metabolites from the secretome hold promise as biomarkers of drug resistance or innovative approaches for cancer treatment, either as standalone therapies or in combination with other medicines.

ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

The pleckstrin homology domain adaptor protein Bam32/DAPP1 is required for germinal center progression.

Ab affinity maturation within germinal centers (GCs) requires weeks to complete. Several signaling pathways in B cells have been shown to be required for initiation of the GC response; however, the signaling checkpoints controlling progression and eventual dissolution of the GC reaction are poorly understood. The adaptor protein Bam32/DAPP1 was originally isolated from human GCs and functions downstream of phosphoinositide 3-kinase enzymes, which are known to have critical roles in B cell activation and GC responses. In this study we identify a unique role of Bam32/DAPP1 in promoting GC progression. Bam32-deficient mice show normal GC initiation, but premature GC dissolution after immunization with protein Ag in alum or low doses of sheep red blood cells. Adoptive transfer studies confirmed that Bam32-deficient B cells have an intrinsic impairment in the ability to mount sustained GC responses. Bam32 deficiency was also associated with impaired Ab affinity maturation. Proliferation of Bam32-deficient GC B cells was not compromised; however, these cells show impaired switch to IgG1 and increased apoptosis in situ. GCs formed by Bam32-deficient B cells contain fewer T cells, indicating that Bam32 is required for B cell-dependent T cell accumulation within established GCs. Exogenous CD40 ligand restored GC B cell numbers and switch to IgG1, indicating that Bam32-deficient B cells are competent to respond to CD40 stimulation when ligand is available. These data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival.

Bam32/DAPP1 promotes B cell adhesion and formation of polarized conjugates with T cells.

B cell Ag receptors function in both signaling activation of Ag-specific cells and in collecting specific Ag for presentation to T lymphocytes. Signaling via PI3K is required for BCR-mediated activation and Ag presentation functions; however, the relevant downstream targets of PI3K in B cells are incompletely defined. In this study, we have investigated the roles of the PI3K effector molecule Bam32/DAPP1 in BCR signaling and BCR-mediated Ag presentation functions. In mouse primary B cells, Bam32 was required for efficient activation of the GTPase Rac1 and downstream signaling to JNK, but not activation of BLNK, phospholipase C gamma2, or calcium responses. Consistent with a role of this adaptor in Rac-mediated cytoskeletal rearrangement, Bam32 was required for BCR-induced cell adhesion and spreading responses on ICAM-1 or fibronectin-coated surfaces. The function of Bam32 in promoting Rac activation and adhesion required tyrosine 139, a known site of phosphorylation by Lyn kinase. After BCR crosslinking by Ag, Bam32-deficient B cells are able to carry out the initial steps of Ag endocytosis and processing, but show diminished ability to form Ag-specific conjugates with T cells and polarize F-actin at the B-T interface. As a result, Bam32-deficient B cells were unable to efficiently activate Ag-specific T cells. Together, these results indicate that Bam32 serves to integrate PI3K and Src kinase signaling to promote Rac-dependent B cell adhesive interactions important for Ag presentation function.

Comprehensive Transcriptome and Pathway Analyses Revealed Central Role for Fascin in Promoting Triple-Negative Breast Cancer Progression.

Recent years have witnessed major progress in development of novel therapeutic agents such as chemotherapy, targeted therapy and immune checkpoint inhibitors for breast cancer. However, cancer-related death remains high especially in triple-negative breast cancer (TNBC) due limited therapeutic options. Development of targeted therapies for TNBC requires better understanding of biology and signaling networks that promote disease progression. Fascin, an actin bundling protein, was identified as a key regulator of many signaling pathways that contribute to breast cancer progression. Herein, fascin ShRNA was used to generate stable fascin knockdown (FSCN1KD) in the MDA-MB-231 TNBC cell line and then were subjected to comprehensive mRNA and miRNA transcriptome analysis. We identified 129 upregulated and 114 downregulated mRNA transcripts, while 14 miRNAs were differentially expressed in FSCN1KD. Ingenuity pathway analysis (IPA) was used to predict the impact of differentially expressed transcripts on signaling pathways and functional categories and to construct miRNA-mRNA regulatory networks in the context of FSCN1 knockdown. Compared to FSCN1KD, fascin-positive (FSCN1CON) breast cancer cells showed enrichment in genes promoting cellular proliferation, migration, survival, DNA replication and repair. Expression of FSCN1high (identified in BRCA dataset from TCGA) in conjunction with elevated expression of the top 10 upregulated or decreased expression of the top 10 downregulated genes (identified in our FSCN1CON vs. FSCN1KD) correlates with worst survival outcome. Taken together, these data confirmed fascin's role in promoting TNBC progression, and identified a novel opportunity for therapeutic interventions via targeting those FSCN1-related transcripts.

Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues.

PURPOSE: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. METHODS: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. RESULTS: In FFPE tissues, PD-L1 was positive in 7-10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20-30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13-20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. CONCLUSIONS: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.