Naser Jafari

Epigenetic Modifications and Therapy in Multiple Sclerosis

Breakthroughs in genetic studies, like whole human genome sequencing and genome-wide association studies (GWAS), have richened our knowledge of etiopathology of autoimmune diseases (AID) through discovery of genetic patterns. Nonetheless, the precise etiology of autoimmune diseases remains largely unknown. The lack of complete concordance of autoimmune disease in identical twins suggests that non-genetic factors also play a major role in determining disease susceptibility. Although there is no certain definition, epigenetics has been known as heritable alterations in gene function without changes in the nucleotide sequence. DNA methylation, histone modifications, and microRNA-associated gene expression suppression are the central mechanisms for epigenetic regulations. Multiple sclerosis (MS) is a disorder of the central nervous system (CNS), characterized by both inflammatory and neurodegenerative features. Although studies on epigenetic alterations in MS only began in the past decade, a mounting number of surveys suggest that epigenetic changes may be involved in the initiation and development of MS, probably through bridging the effects of environmental risk factors to genetics. Arming with clear understanding of epigenetic dysregulations underpins development of epigenetic therapies. Identifying agents inhibiting the enzymes controlling epigenetic modifications, particularly DNA methyltransferases and histone deacetylases, will be promising therapeutic tool toward MS. In the article underway, it is aimed to go through the recent progresses, attempting to disclose how epigenetics associates with the pathogenesis of MS and how can be used as therapeutic approach.

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

Talin2-mediated traction force drives matrix degradation and cell invasion.

ABSTRACT Talin binds to β-integrin tails to activate integrins, regulating cell migration, invasion and metastasis. There are two talin genes, TLN1 and TLN2, encoding talin1 and talin2, respectively. Talin1 regulates focal adhesion dynamics, cell migration and invasion, whereas the biological function of talin2 is not clear and, indeed, talin2 has been presumed to function redundantly with talin1. Here, we show that talin2 has a much stronger binding to β-integrin tails than talin1. Replacement of talin2 Ser339 with Cys significantly decreased its binding to β1-integrin tails to a level comparable to that of talin1. Talin2 localizes at invadopodia and is indispensable for the generation of traction force and invadopodium-mediated matrix degradation. Ablation of talin2 suppressed traction force generation and invadopodia formation, which were restored by re-expressing talin2 but not talin1. Furthermore, re-expression of wild-type talin2 (but not talin2S339C) in talin2-depleted cells rescued development of traction force and invadopodia. These results suggest that a strong interaction of talin2 with integrins is required to generate traction, which in turn drives invadopodium-mediated matrix degradation, which is key to cancer cell invasion. Summary: Talin2, previously presumed to function redundantly with talin1, binds to β integrins more strongly than talin1, and generates traction force to regulate invadopodium formation and cell invasion.

Potent Anti-Cancer Effects of Less Polar Curcumin Analogues on Gastric Adenocarcinoma and Esophageal Squamous Cell Carcinoma Cells

Curcumin and its chalcone derivatives inhibit the growth of human cancer cells. It is reported that replacement of two OH groups in curcumin with less polar groups like methoxy increases its anti-proliferative activity. In this study, we explored benzylidine cyclohexanone derivatives with non-polar groups, to see if they possess increased anti-cancer activity. Novel 2,6-bis benzylidine cyclohexanone analogues of curcumin were synthesized, and their inhibitory effects on gastric adenocarcinoma (AGS) and esophageal squamous cell carcinoma (KYSE30) cancer cells were studied using an MTT assay. Cell apoptosis was detected by EB/AO staining, and cell cycle was analyzed by flow cytometry. Real-time PCR was performed for gene expression analysis. All synthesized analogues were cytotoxic toward gastric and esophageal cancer cells and showed lower IC50 values than curcumin. Treatment with 2,6-Bis-(3-methoxy-4-propoxy-benzylidene)-cyclohexanone (BM2) was 17 times more toxic than curcumin after 48 h incubation. All novel compounds were more effective than curcumin in apoptosis induction and cell cycle arrest at G1 phase. These results suggest that less polar analogues of curcumin have potent cytotoxicity in vitro. However, they need to be investigated further, especially with animal tumor models, to confirm their chemotherapeutic activity in vivo.

p70S6K1 (S6K1)-mediated Phosphorylation Regulates Phosphatidylinositol 4-Phosphate 5-Kinase Type I γ Degradation and Cell Invasion

Phosphatidylinositol 4-phosphate 5-kinase type I γ (PIPKIγ90) ubiquitination and subsequent degradation regulate focal adhesion assembly, cell migration, and invasion. However, it is unknown how upstream signals control PIPKIγ90 ubiquitination or degradation. Here we show that p70S6K1 (S6K1), a downstream target of mechanistic target of rapamycin (mTOR), phosphorylates PIPKIγ90 at Thr-553 and Ser-555 and that S6K1-mediated PIPKIγ90 phosphorylation is essential for cell migration and invasion. Moreover, PIPKIγ90 phosphorylation is required for the development of focal adhesions and invadopodia, key machineries for cell migration and invasion. Surprisingly, substitution of Thr-553 and Ser-555 with Ala promoted PIPKIγ90 ubiquitination but enhanced the stability of PIPKIγ90, and depletion of S6K1 also enhanced the stability of PIPKIγ90, indicating that PIPKIγ90 ubiquitination alone is insufficient for its degradation. These data suggest that S6K1-mediated PIPKIγ90 phosphorylation regulates cell migration and invasion by controlling PIPKIγ90 degradation.

The role of talin2 in breast cancer tumorigenesis and metastasis

Recent studies show that talin2 has a higher affinity to β-integrin tails and is indispensable for traction force generation and cell invasion. However, its roles in cell migration, cancer cell metastasis and tumorigenesis remain to be determined. Here, we used MDA-MB-231 human breast cancer cells as a model to define the roles of talin2 in cell migration, invasion, metastasis and tumorigenesis. We show here that talin2 knockdown (KD) inhibited cell migration and focal adhesion dynamics, a key step in cell migration, and that talin2 knockout (KO) inhibited cell invasion and traction force generation, the latter is crucial for cell invasion. Re-expression of talin2WT in talin2-KO cells restored traction force generation and cell invasion, but that of talin2S339C, a β-integrin-binding deficient mutant, did not. Moreover, talin2 KO (or KD) suppressed tumorigenesis and metastasis in mouse xenograft models. However, surprisingly, re-expression of talin2WT in talin2-KO cells did not rescue tumorigenesis. Thus, talin2 is required for breast cancer cell migration, invasion, metastasis and tumorigenesis, although exogenous expression of high levels of talin2 could inhibit tumorigenesis.

Cdk5-mediated phosphorylation regulates phosphatidylinositol 4-phosphate 5-kinase type I γ 90 activity and cell invasion

Phosphatidylinositol 4‐phosphate 5‐kinase type I γ (PIPKIγ90) regulates cell migration, invasion, and metastasis. However, it is unknown how cellular signals regulate those processes. Here, we show that cyclin‐dependent kinase 5 (Cdk5), a protein kinase that regulates cell migration and invasion, phosphorylates PIPKIγ90 at S453, and that Cdk5‐mediated PIPKIγ90 phosphorylation is essential for cell invasion. Moreover, Cdk5‐mediated phosphorylation down‐regulates the activity of PIPKIγ90 and the secretion of fibronectin, an extracellular matrix protein that regulates cell migration and invasion. Furthermore, inhibition of PIPKIγ activity with the chemical inhibitor UNC3230 suppresses fibronectin secretion in a dose‐dependent manner, whereas depletion of Cdk5 enhances fibronectin secretion. With total internal reflection fluorescence microscopy, we found that secreted fibronectin appears as round dots, which colocalize with Tks5 and CD9 but not with Zyxin. These data suggest that Cdk5‐mediated PIPKIγ90 phosphorylation regulates cell invasion by controlling PIPKIγ90 activity and fibronectin secretion.—Li, L., Kotodziej, T., Jafari, N., Chen, J., Zhu, H., Rajfur, Z., Huang, C. Cdk5‐mediated phosphorylation regulates phosphatidylinositol 4‐phosphate 5‐kinase type I γ 90 activity and cell invasion. FASEB J. 33, 631–642 (2019). www.fasebj.org

De Novo Fatty Acid Synthesis-Driven Sphingolipid Metabolism Promotes Metastatic Potential of Colorectal Cancer

Metastasis is the most common cause of death in colorectal cancer patients. Fatty acid synthase (FASN) and sphingosine kinase-1 and -2 (SPHK1 and 2) are overexpressed in many cancers, including colorectal cancer. However, the contribution of FASN-mediated upregulation of sphingolipid metabolism to colorectal cancer metastasis and the potential of these pathways as targets for therapeutic intervention remain unknown. This study determined that sphingosine kinases (SPHK) are overexpressed in colorectal cancer as compared with normal mucosa. FASN expression significantly correlated with SPHK2 expression in data sets from The Cancer Genome Atlas (TCGA) and a colorectal cancer tumor microarray. FASN, SPHK1, and SPHK2 colocalized within invadopodia of primary colorectal cancer cells. Moreover, FASN inhibition decreased SPHK2 expression and the levels of dihydrosphingosine 1-phosphate (DH-S1P) and sphingosine 1-phosphate (S1P) in colorectal cancer cells and tumor tissues. Inhibition of FASN using TVB-3664 and sphingolipid metabolism using FTY-720 significantly inhibited the ability of primary colorectal cancer cells to proliferate, migrate, form focal adhesions, and degrade gelatin. Inhibition of the FASN/SPHK/S1P axis was accompanied by decreased activation of p-MET, p-FAK, and p-PAX. S1P treatment rescued FASN-mediated inhibition of these proteins, suggesting that FASN promotes metastatic properties of colorectal cancer cells, in part, through an increased sphingolipid metabolism. These data demonstrate that upregulation of the FASN/SPHK/S1P axis promotes colorectal cancer progression by enhancing proliferation, adhesion, and migration. Implications: This study provides a strong rationale for further investigation of the interconnection of de novo lipogenesis and sphingolipid metabolism that could potentially lead to the identification of new therapeutic targets and strategies for colorectal cancer.

Overexpression of Drosha, DiGeorge syndrome critical region gene 8 (DGCR8), and Dicer mRNAs in the pathogenesis of psoriasis

Psoriasis is a complex autoimmune inflammatory disease that occurs in genetically susceptible individuals and presents with the development of inflammatory plaques on the skin. Recent studies have indicated that microRNAs (miRNAs) play important roles in psoriasis.