pVHL Acts as an Adaptor to Promote the Inhibitory Phosphorylation of the NF-κB Agonist Card9 by CK2

The VHL tumor suppressor protein (pVHL) is part of an E3 ubiquitin ligase that targets HIF for destruction. pVHL-defective renal carcinoma cells exhibit increased NF-κB activity but the mechanism is unclear. NF-κB affects tumorigenesis and therapeutic resistance in some settings. We found that pVHL associates with the NF-κB agonist Card9 but does not target Card9 for destruction. Instead, pVHL serves as an adaptor that promotes the phosphorylation of the Card9 C-terminus by CK2. Elimination of these sites markedly enhanced Card9's ability to activate NF-κB in VHL+/+ cells and Card9 siRNA normalized NF-κB activity in VHL−/− cells and restored their sensitivity to cytokine-induced apoptosis. Furthermore, downregulation of Card9 in VHL−/− cancer cells reduced their tumorigenic potential. Therefore pVHL can serve as an adaptor for both an ubiquitin conjugating enzyme and for a kinase. The latter activity, which promotes Card9 phosphorylation, links pVHL to control of NF-κB activity and tumorigenesis

Interaction of YY1 with E2Fs, mediated by RYBP, provides a mechanism for specificity of E2F function

To explore mechanisms for specificity of function within the family of E2F transcription factors, we have identified proteins that interact with individual E2F proteins. A two-hybrid screen identified RYBP (Ring1- and YY1-binding protein) as a protein that interacts specifically with the E2F2 and E2F3 family members, dependent on the marked box domain in these proteins. The Cdc6 promoter contains adjacent E2F- and YY1-binding sites, and both are required for promoter activity. In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, can stimulate Cdc6 promoter activity synergistically, dependent on the marked box domain of E2F3. Using chromatin immunoprecipitation assays, we show that both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at G(1)/S of the cell cycle. In contrast, we detect no interaction of E2F1 with the Cdc6 promoter. We suggest that the ability of RYBP to mediate an interaction between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and provides a basis for specificity of E2F function

The 2-oxoglutarate analog 3-oxoglutarate decreases normoxic hypoxia-inducible factor-1α in cancer cells, induces cell death, and reduces tumor xenograft growth

The cellular response to hypoxia is primarily regulated by the hypoxia-inducible factors (HIFs). HIF-1α is also a major mediator of tumor physiology, and its abundance is correlated with therapeutic resistance in a broad range of cancers. Accumulation of HIF-1α under hypoxia is mainly controlled by the oxygen-sensing HIF prolyl 4-hydroxylases (EGLNs, also known as PHDs). Here, we identified a high level of normoxic HIF-1α protein in various cancer cell lines. EGLNs require oxygen and 2-oxoglutarate for enzymatic activity. We tested the ability of several cell-permeable 2-oxoglutarate analogs to regulate the abundance of HIF-1α protein. We identified 3-oxoglutarate as a potent regulator of HIF-1α in normoxic conditions. In contrast to 2-oxoglutarate, 3-oxoglutarate decreased the abundance of HIF-1α protein in several cancer cell lines in normoxia and diminished HIF-1α levels independent of EGLN enzymatic activity. Furthermore, we observed that 3-oxoglutarate was detrimental to cancer cell survival. We show that esterified 3-oxoglutarate, in combination with the cancer chemotherapeutic drug vincristine, induces apoptosis and inhibits tumor growth in vitro and in vivo. Our data imply that a novel treatment strategy targeting HIF-1α in combination with the use of existing cytotoxic agents could serve as potent, future antitumor chemotherapies

CD25bright NK cells display superior function and metabolic activity under regulatory T cell-mediated suppression

ABSTRACTInfusion of natural killer (NK) cells is an attractive therapeutic modality in patients with cancer. However, the activity of NK cells is regulated by several mechanisms operating within solid tumors. Regulatory T (Treg) cells suppress NK cell activity through various mechanisms including deprivation of IL-2 via the IL-2 receptor alpha (CD25). Here, we investigate CD25 expression on NK cells to confer persistence in Treg cells containing solid tumor models of renal cell carcinoma (RCC). Compared with IL-2, stimulation with IL-15 increases the expression of CD25 resulting in enhanced response to IL-2 as evidenced by increased phosphorylation of STAT5. Compared with CD25dim NK cells, CD25bright NK cells isolated from IL-15 primed NK cells display increased proliferative and metabolic activity as well as increased ability to persist in Treg cells containing RCC tumor spheroids. These results support strategies to enrich for or selectively expand CD25bright NK cells for adoptive cellular therapy of NK cells

A role for E2F6 in distinguishing G1/S- and G2/M-specific transcription

E2F transcription factors play a critical role in the control of cell cycle progression, regulating the expression of genes involved in DNA replication, DNA repair, mitosis, and cell fate. This involves both positive-acting and negative-acting E2F proteins, the latter group including the E2F6 protein, which has been shown to function as an Rb-independent repressor of E2F-target gene transcription. In an effort to better delineate the context of E2F6 function, including the mechanisms of E2F6 functional specificity, we used chromatin immunoprecipitation assays to assess when and with what genes E2F6 associates during a cell cycle. We find that E2F6 associates specifically with the E2F target genes that are activated at G1/S; this interaction occurs during S phase of the cell cycle. In sharp contrast, E2F6 does not bind to E2F-regulated genes activated at G2/M. In the absence of E2F6, E2F4 can bind to the G1/S-regulated promoters and compensate for loss of E2F6 function. Indeed, inhibition of both E2F4 and E2F6 activity results in specific derepression of these genes during S phase. We conclude that E2F6 functions as a repressor of E2F-dependent transcription during S phase and given the specificity for the G1/S-regulated genes, we propose that E2F6 functions to distinguish G1/S and G2/M transcription during the cell cycle

Platelet factor 4 enhances CD4(+)T effector memory cell responses via Akt-PGC1 alpha-TFAM signaling-mediated mitochondrial biogenesis

Background Cell metabolism drives T cell functions, while platelets regulate overall CD4(+)T cell immune responses. Objective To investigate if platelets influence cell metabolism and thus regulate CD4(+)T effector memory cell (Tem) responses. Methods Human CD4(+)Tem cells were activated with alpha CD3/alpha CD28 and cultured without or with platelets or platelet-derived mediators. Results Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4(+)Tem cells. Platelet co-culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet-derived mediator regulating CD4(+)Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1 alpha phosphorylation, and resulted in elevations of PGC1 alpha function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM-adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T-bet) and Treg (FoxP3) transcription factor expression and corresponding CD4(+)T effector cell responses. Conclusions Platelets enhance CD4(+)T cell responses of Tem cells through PF4-dependent and Akt-PGC1 alpha-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses