Abdol Hossein Rezaeian

Posttranslational regulation of Akt in human cancer

Akt regulates critical cellular processes including cell survival and proliferation, glucose metabolism, cell migration, cancer progression and metastasis through phosphorylation of a variety of downstream targets. The Akt pathway is one of the most prevalently hyperactivated signaling pathways in human cancer, thus, research deciphering molecular mechanisms which underlie the aberrant Akt activation has received enormous attention. The PI3K-dependent Akt serine/threonine phosphorylation by PDK1 and mTORC2 has long been thought to be the primary mechanism accounting for Akt activation. However, this regulation alone does not sufficiently explain how Akt hyperactivation can occur in tumors with normal levels of PI3K/PTEN activity. Mounting evidence demonstrates that aberrant Akt activation can be attributed to other posttranslational modifications, which include tyrosine phosphorylation, O-GlcNAcylation, as well as lysine modifications: ubiquitination, SUMOylation and acetylation. Among them, K63-linked ubiquitination has been shown to be a critical step for Akt signal activation by facilitating its membrane recruitment. Deficiency of E3 ligases responsible for growth factor-induced Akt activation leads to tumor suppression. Therefore, a comprehensive understanding of posttranslational modifications in Akt regulation will offer novel strategies for cancer therapy.

Skp2 E3 Ligase Integrates ATM Activation and Homologous Recombination Repair by Ubiquitinating NBS1

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.

Skp2-Mediated RagA Ubiquitination Elicits a Negative Feedback to Prevent Amino-Acid-Dependent mTORC1 Hyperactivation by Recruiting GATOR1

The regulation of RagA(GTP) is important for amino-acid-induced mTORC1 activation. Although GATOR1 complex has been identified as a negative regulator for mTORC1 by hydrolyzing RagA(GTP), how GATOR1 is recruited to RagA to attenuate mTORC1 signaling remains unclear. Moreover, how mTORC1 signaling is terminated upon amino acid stimulation is also unknown. We show that the recruitment of GATOR1 to RagA is induced by amino acids in an mTORC1-dependent manner. Skp2 E3 ligase drives K63-linked ubiquitination of RagA, which facilitates GATOR1 recruitment and RagA(GTP) hydrolysis, thereby providing a negative feedback loop to attenuate mTORC1 lysosomal recruitment and prevent mTORC1 hyperactivation. We further demonstrate that Skp2 promotes autophagy but inhibits cell size and cilia growth through RagA ubiquitination and mTORC1 inhibition. We thereby propose a negative feedback whereby Skp2-mediated RagA ubiquitination recruits GATOR1 to restrict mTORC1 signaling upon sustained amino acid stimulation, which serves a critical mechanism to maintain proper cellular functions.

Skp2-MacroH2A1-CDK8 axis orchestrates G2/M transition and tumorigenesis

Understanding the mechanism by which cell growth, migration, polyploidy, and tumourigenesis are regulated may provide important therapeutic strategies for cancer therapy. Here we identify the Skp2-macroH2A1 (mH2A1)-CDK8 axis as a critical pathway for these processes, and deregulation of this pathway is associated with human breast cancer progression and patient survival outcome. We showed that mH2A1 is a new substrate of Skp2 SCF complex whose degradation by Skp2 promotes CDK8 gene and protein expression. Strikingly, breast tumour suppression upon Skp2 deficiency can be rescued by mH2A1 knockdown or CDK8 restoration using mouse tumour models. We further show that CDK8 regulates p27 protein expression by facilitating Skp2-mediated p27 ubiquitination and degradation. Our study establishes a critical role of Skp2-mH2A1-CDK8 axis in breast cancer development and targeting this pathway offers a promising strategy for breast cancer therapy.

Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex

Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability. Finally, we show that the oncogenic activity of Skp2 is repressed by Foxo3a overexpression. Our results not only reveal novel insights into how Skp2 SCF complex is regulated, but also establish a new role for Foxo3a in tumor suppression through a transcription-dependent and independent manner.

A hypoxia-responsive TRAF6–ATM–H2AX signalling axis promotes HIF1α activation, tumorigenesis and metastasis

The understanding of how hypoxia stabilizes and activates HIF1α in the nucleus with related oncogenic signals could revolutionize targeted therapy for cancers. Here, we find that histone H2AX displays oncogenic activity by serving as a crucial regulator of HIF1α signalling. H2AX interacts with HIF1α to prevent its degradation and nuclear export in order to allow successful VHL-independent HIF1α transcriptional activation. We show that mono-ubiquitylation and phosphorylation of H2AX, which are strictly mediated by hypoxia-induced E3 ligase activity of TRAF6 and ATM, critically regulate HIF1α-driven tumorigenesis. Importantly, TRAF6 and γH2AX are overexpressed in human breast cancer, correlate with activation of HIF1α signalling, and predict metastatic outcome. Thus, TRAF6 and H2AX overexpression and γH2AX-mediated HIF1α enrichment in the nucleus of cancer cells lead to overactivation of HIF1α-driven tumorigenesis, glycolysis and metastasis. Our findings suggest that TRAF6-mediated mono-ubiquitylation and subsequent phosphorylation of H2AX may serve as potential means for cancer diagnosis and therapy.

Atad3a suppresses Pink1-dependent mitophagy to maintain homeostasis of hematopoietic progenitor cells article

Although deletion of certain autophagy-related genes has been associated with defects in hematopoiesis, it remains unclear whether hyperactivated mitophagy affects the maintenance and differentiation of hematopoietic stem cells (HSCs) and committed progenitor cells. Here we report that targeted deletion of the gene encoding the AAA+-ATPase Atad3a hyperactivated mitophagy in mouse hematopoietic cells. Affected mice showed reduced survival, severely decreased bone-marrow cellularity, erythroid anemia and B cell lymphopenia. Those phenotypes were associated with skewed differentiation of stem and progenitor cells and an enlarged HSC pool. Mechanistically, Atad3a interacted with the mitochondrial channel components Tom40 and Tim23 and served as a bridging factor to facilitate appropriate transportation and processing of the mitophagy protein Pink1. Loss of Atad3a caused accumulation of Pink1 and activated mitophagy. Notably, deletion of Pink1 in Atad3a-deficient mice significantly ‘rescued’ the mitophagy defect, which resulted in restoration of the progenitor and HSC pools. Our data indicate that Atad3a suppresses Pink1-dependent mitophagy and thereby serves a key role in hematopoietic homeostasis.The survival of hematopoietic stem cells requires tight regulation of mitophagy. Lin and colleagues show that Atad3a regulates mitophagy in these cells by sequestering the mitophagy initiator Pink1 and directing its import via the mitochondrial Tom40–Tim23 complex.

CD74 interferes with the expression of fas receptor on the surface of lymphoma cells

BackgroundResistance to Fas-mediated apoptosis limits the efficacy of currently available chemotherapy regimens. We identified CD74, which is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.MethodsCD74 expression was suppressed in human B-lymphoma cell lines, BJAB and Raji, by either transduction with lentivirus particles or transfection with episomal vector, both encoding CD74-specific shRNAs or non-target shRNA. Effect of CD74 expression on Fas signaling was evaluated by comparing survival of mice hydrodynamically transfected with vector encoding full-length CD74 or empty vector. Sensitivity of cells with suppressed CD74 expression to FasL, edelfosine, doxorubicin, and a humanized CD74-specific antibody, milatuzumab, was evaluated by flow cytometry and compared to control cells. Fas signaling in response to FasL stimulation and the expression of Fas signaling components were evaluated by Western blot. Surface expression of Fas was detected by flow cytometry.ResultsWe determined that cells with suppressed CD74 are more sensitive to FasL-induced apoptosis and Fas signaling-dependent chemotherapies, edelfosine and doxorubicin, than control CD74-expressing cells. On the other hand, expression of full-length CD74 in livers protected the mice from a lethal challenge with agonistic anti-Fas antibody Jo2. A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74, suggesting that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. Cells with suppressed CD74 expression showed increased staining of Fas receptor on their surface. Pre-treatment with milatuzumab sensitized BJAB cells to Fas-mediated apoptosis.ConclusionWe anticipate that specific targeting of the CD74 on the cell surface will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis, and thus will increase effectiveness of chemotherapy regimens for hematological malignancies.

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo.

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

H3 ubiquitination by NEDD4 regulates H3 acetylation and tumorigenesis

Dynamic changes in histone modifications under various physiological cues play important roles in gene transcription and cancer. Identification of new histone marks critical for cancer development is of particular importance. Here we show that, in a glucose-dependent manner, E3 ubiquitin ligase NEDD4 ubiquitinates histone H3 on lysine 23/36/37 residues, which specifically recruits histone acetyltransferase GCN5 for subsequent H3 acetylation. Genome-wide analysis of chromatin immunoprecipitation followed by sequencing reveals that NEDD4 regulates glucose-induced H3 K9 acetylation at transcription starting site and enhancer regions. Integrative analysis of ChIP-seq and microarray data sets also reveals a consistent role of NEDD4 in transcription activation and H3 K9 acetylation in response to glucose. Functionally, we show that NEDD4-mediated H3 ubiquitination, by transcriptionally activating IL1α, IL1β and GCLM, is important for tumour sphere formation. Together, our study reveals the mechanism for glucose-induced transcriptome reprograming and epigenetic regulation in cancer by inducing NEDD4-dependent H3 ubiquitination.