Musaad Alfayez

Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers

Effective management of breast cancer depends on early diagnosis and proper monitoring of patients’ response to therapy. However, these goals are difficult to achieve because of the lack of sensitive and specific biomarkers for early detection and for disease monitoring. Accumulating evidence in the past several years has highlighted the potential use of peripheral blood circulating nucleic acids such as DNA, mRNA and micro (mi)RNA in breast cancer diagnosis, prognosis and for monitoring response to anticancer therapy. Among these, circulating miRNA is increasingly recognized as a promising biomarker, given the ease with which miRNAs can be isolated and their structural stability under different conditions of sample processing and isolation. In this review, we provide current state-of-the-art of miRNA biogenesis, function and discuss the advantages, limitations, as well as pitfalls of using circulating miRNAs as diagnostic, prognostic or predictive biomarkers in breast cancer management.

Characterization of Cellular and Molecular Heterogeneity of Bone Marrow Stromal Cells

Human bone marrow-derived stromal stem cells (hBMSC) exhibit multiple functions, including differentiation into skeletal cells (progenitor function), hematopoiesis support, and immune regulation (nonprogenitor function). We have previously demonstrated the presence of morphological and functional heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development and osteoblast differentiation genes which included several homeobox genes: TBX15, HOXA2 and HOXA10, and IGF1, FGFR3, BMP6, MCAM, ITGA10, IGFBP5, and ALP. siRNA-based downregulation of the ALP gene in CL1 impaired osteoblastic and adipocytic differentiation. Our studies demonstrate the existence of molecular and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures.

CUDC-907 Promotes Bone Marrow Adipocytic Differentiation Through Inhibition of Histone Deacetylase and Regulation of Cell Cycle

The role of bone marrow adipocytes (BMAs) in overall energy metabolism and their effects on bone mass are currently areas of intensive investigation. BMAs differentiate from bone marrow stromal cells (BMSCs); however, the molecular mechanisms regulating BMA differentiation are not fully understood. In this study, we investigated the effect of CUDC-907, identified by screening an epigenetic small-molecule library, on adipocytic differentiation of human BMSCs (hBMSCs) and determined its molecular mechanism of action. Human bone marrow stromal cells exposed to CUDC-907 (500 nM) exhibited enhanced adipocytic differentiation (∼2.9-fold increase, P < 0.005) compared with that of control cells. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis, cell cycle, and DNA replication. Chromatin immune precipitation combined with quantitative polymerase chain reaction showed significant increase in H3K9ac epigenetic marker in the promoter regions of AdipoQ, FABP4, PPARγ, KLF15, and CEBPA in CUDC-907-treated hBMSCs. Follow-up experiments corroborated that the inhibition of histone deacetylase (HDAC) activity enhanced adipocytic differentiation, while the inhibition of PI3K decreased adipocytic differentiation. In addition, CUDC-907 arrested hBMSCs in the G0-G1 phase of the cell cycle and reduced the number of S-phase cells. Our data reveal that HDAC, PI3K, and cell cycle genes are important regulators of BMA formation and demonstrate that adipocyte differentiation of hBMSCs is associated with complex changes in a number of epigenetic and genetic pathways, which can be targeted to regulate BMA formation.

Rapid Biological Synthesis of Silver Nanoparticles Using Plant Seed Extracts and Their Cytotoxicity on Colorectal Cancer Cell Lines

In this research we focused on the green synthesis of silver nanoparticles (AgNPs) using Pimpinella anisum seed extract. Furthermore, we evaluated their cytotoxicity on colorectal cancer (CRC) cell lines. Our results revealed the anti-cancerous cytotoxic potential of green synthesized AgNPs. Green synthesized AgNPs exhibited high cytotoxicity on colorectal adenocarcinoma CRC cells. They selectively killed cancer cells through suppression of proliferation, cell cycle arrest at the G2/M phase, and induction of apoptosis. Interestingly, between the two different clones, SW620 cells were more sensitive than HCT8 cells. Overall, our findings suggest that AgNPs could be effective cancer chemotherapeutic agents or a combination nano-drug in future anti-cancer therapy.

Inhibitory Effects of Toll-Like Receptor 4, NLRP3 Inflammasome, and Interleukin-1β on White Adipocyte Browning

Adipose tissue expansion is accompanied by infiltration and accumulation of pro-inflammatory macrophages, which links obesity to pathologic conditions such as type 2 diabetes. However, little is known regarding the role of pro-inflammatory adipose tissue remodeling in the thermogenic activation of brown/beige fat. Here, we investigated the effect of pattern recognition receptors (PRR) activation in macrophages, especially the toll-like receptor 4 (TLR4) and Nod-like receptor 3 (NLRP3), on white adipocyte browning. We report that TLR4 activation by lipopolysaccharide repressed white adipocyte browning in response to β3-adrenergic receptor activation and caused ROS production and mitochondrial dysfunction, while genetic deletion of TLR4 protected mitochondrial function and thermogenesis. In addition, activation of NLRP3 inflammasome in macrophages attenuated UCP1 induction and mitochondrial respiration in cultures of primary adipocytes, while the absence of NLRP3 protected UCP1 in adipocytes. The effect of NLRP3 inflammasome activation on browning was mediated by IL-1β signaling, as blocking IL-1 receptor in adipocytes protected thermogenesis. We also report that IL-1β interferes with thermogenesis via oxidative stress stimulation and mitochondrial dysfunction as we observed a statistically significant increase in ROS production, decrease in SOD enzyme activity, and increase in mitochondrial depolarization in adipocytes treated with IL-1β. Collectively, we demonstrated that inflammatory response to obesity, such as TLR4 and NLRP3 inflammasome activation as well as IL-1β secretion, attenuates β3-adrenoreceptor-induced beige adipocyte formation via oxidative stress and mitochondrial dysfunction. Our findings provide insights into targeting innate inflammatory system for enhancement of the adaptive thermogenesis against obesity.

Molecular profiling of ALDH1 + colorectal cancer stem cells reveals preferential activation of MAPK, FAK, and oxidative stress prosurvival signalling pathways

Tumour heterogeneity leads to variable clinical response and inaccurate diagnostic and prognostic assessment. Cancer stem cells (CSCs) represent a subpopulation responsible for invasion, metastasis, therapeutic resistance, and recurrence in many human cancer types. However, the true identity of colorectal cancer (CRC) SCs remains elusive. Here, we aimed to characterize and define the gene expression portrait of CSCs in CRC-model SW403 cells. We found that ALDH+ positive cells are clonogenic and highly proliferative; their global gene expression profiling-based molecular signature revealed gene enrichment related to DNA damage, MAPK, FAK, oxidative stress response, and Wnt signalling. ALDH+ cells showed enhanced ROS stress resistance, whereas MAPK/FAK pathway pharmacologic inhibition limited their survival. Conversely, 5-fluorouracil increased the ALDH+ cell fraction among the SW403, HCT116 and SW620 CRC models. Notably, analysis of ALDH1A1 and POU5F1 expression levels in cohorts of 462 or 420 patients for overall (OS) or disease-free (DFS) survival, respectively, obtained from the Cancer Genome Atlas CRC dataset, revealed strong association between elevated expression and poor OS (p = 0.006) and poor DFS (p = 0.05), thus implicating ALDH1A1 and POU5F1 in CRC prognosis. Our data reveal distinct molecular signature of ALDH+ CSCs in CRC and suggest pathways relevant for successful targeted therapies and management of CRC.

Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells

Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand, pathway analysis on the down-regulated genes revealed significant enrichment in pathways related to cell cycle regulation. Based on these data, we assessed the effect of pharmacological inhibition of FAK signaling using PF-573228, PF-562271, and InsR/IGF-1R using NVP-AEW541 and GSK-1904529A on adipocyte differentiation. hMSCs exposed to FAK or IGF-1R/InsR inhibitors exhibited fewer adipocyte formation (27–58% inhibition, P<0005). Concordantly, the expression of adipocyte-specific genes AP2, AdipoQ, and CEBPα was significantly reduced. On the other hand, we did not detect significant effects on cell viability as a result of FAK or IGF-1R/InsR inhibition. Our data identified FAK and insulin signaling as important intracellular signaling pathways relevant to bone marrow adipogenesis.

TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton

TGFβ is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGFβ1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGFβ-mediated enhancement of lineage commitment, we compared the gene expression profiles of TGFβ1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGFβl treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGFβ1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGFβ1 and cytochalasin D. Our study demonstrates that TGFβ1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity

Bone marrow mesenchymal stem cells (BMSCs) are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM) exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGFβ signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPARγ) and KLF15 (related to adipogenesis) or SP7 (Osterix) and alkaline phosphatase (ALP) (related to osteogenesis) in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs.

ACI/EG eutectic mixture mediated synthesis, characterization and in vitro osteoblast differentiation assessment of spiropyrrolo[1,2-b]isoquinoline analogues

An eco-friendly acetylcholine iodide–ethylene glycol (ACI/EG) deep eutectic mixture mediated green protocol has been developed for the synthesis of hitherto unexplored multi-functionalized linear tricyclic spiropyrrolo[1,2-b]isoquinoline analogues. The effects of the synthesized compounds on the osteoblast differentiation of hBMSC-TERT cell lines were investigated and promising results were observed with significant IC50 values. In addition, molecular modeling simulations were also performed with the 3D structure of BMP-2 to reveal binding interactions and orientations of highly potent spiropyrrolo[1,2-b]isoquinoline analogues.