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Objective: Vascular endothelial growth factor (VEGF), apart from its predominant roles in angiogenesis, can enhance cancer cellproliferation, but its mechanisms remain elusive. The purpose of the present study was therefore to identify how VEGF regulatescancer cell proliferation. Methods: VEGF effects on cancer cell proliferation were investigated with the VEGF receptor 2 inhibitor, Ki8751, and the breastcancer cell lines, MCF-7 and MDA-MB-231, using flow cytometry, mass spectrometry, immunoblotting, and confocal microscopy.Data were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. Results: VEGF blockade by Ki8751 significantly reduced cancer cell proliferation, and enhanced breast cancer cell apoptosis.Mass spectrometric analyses revealed that Ki8751 treatment significantly upregulated the expression of mitochondrial proteins,suggesting the involvement of mitochondrial biogenesis. Confocal microscopy and flow cytometric analyses showed that Ki8751treatment robustly increased the mitochondrial masses of both cancer cells, induced endomitosis, and arrested cancer cells in the highaneuploid phase. VEGFR2 knockdown by shRNAs showed similar effects to those of Ki8751, confirming the specificity of Ki8751treatment. Enhanced mitochondrial biogenesis increased mitochondrial oxidative phosphorylation and stimulated reactive oxygenspecies (ROS) production, which induced cancer cell apoptosis. Furthermore, Ki8751 treatment downregulated the phosphorylationof Akt and PGC1α, and translocated PGC1α into the nucleus. The PGC1α alterations increased mitochondrial transcription factorA (TFAM) expression and subsequently increased mitochondrial biogenesis. Conclusions: VEGF enhances cancer cell proliferation by decreasing Akt-PGC1α-TFAM signaling-mediated mitochondrialbiogenesis, ROS production, and cell apoptosis. These findings suggested the anticancer potential of Ki8751 via increasedmitochondrial biogenesis and ROS production.

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 αCD3/αCD28 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 PGC1α phosphorylation, and resulted in elevations of PGC1α 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-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel–Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel–Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

We recently identified pathogenic KIF1Bβ mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bβ in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bβ is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bβ mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bβ-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bβ. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bβ independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bβ loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bβ mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Through analysis of RNA-sequenced neuroblastoma we here show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, generating a truncated BAG2-protein which inhibited retinoic acid-induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism producing altered gene products in neuroblastoma and suggest that the spliceosome is a tractable therapeutical target.

KIF1Bβ is a candidate 1p36 tumor suppressor that regulates apoptosis in the developing sympathetic nervous system. We found that KIF1Bβ activates the Ca(2+)-dependent phosphatase calcineurin (CN) by stabilizing the CN-calmodulin complex, relieving enzymatic autoinhibition and enabling CN substrate recognition. CN is the key mediator of cellular responses to Ca(2+) signals and its deregulation is implicated in cancer, cardiac, neurodegenerative, and immune disease. We show that KIF1Bβ affects mitochondrial dynamics through CN-dependent dephosphorylation of Dynamin-related protein 1 (DRP1), causing mitochondrial fission and apoptosis. Furthermore, KIF1Bβ actuates recognition of all known CN substrates, implying a general mechanism for KIF1Bβ in Ca(2+) signaling and how Ca(2+)-dependent signaling is executed by CN. Pathogenic KIF1Bβ mutations previously identified in neuroblastomas and pheochromocytomas all fail to activate CN or stimulate DRP1 dephosphorylation. Importantly, KIF1Bβ and DRP1 are silenced in 1p36 hemizygous-deleted neuroblastomas, indicating that deregulation of calcineurin and mitochondrial dynamics contributes to high-risk and poor-prognosis neuroblastoma.

• Introduction • Lessons from a rare disease • Specificity of function within the EglN prolyl hydroxylases • Failure of EglN3-mediated apoptosis in the genesis of phaeochromocytoma • Understanding the mechanistic basis of EglN3 killing • Connecting EglN activity to the Warburg conundrum • Future directions Oxygen-sensing mechanisms are often dysfunctional in tumours. Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor α (HIF-α) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes (SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor α-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. α-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFα and/or resistance to EglN3-mediated apoptosis.

Germline von Hippel-Lindau tumour suppressor gene (VHL) mutations cause renal cell carcinomas, haemangioblastomas and phaeochromocytomas in humans. Mutations in VHL also occur in sporadic renal cell carcinomas. The protein encoded by VHL, VHL, is part of the ubiquitin ligase that downregulates the heterodimeric transcription factor Hif under well-oxygenated conditions. Here we show that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo. This phenotype was independent of p53 and Hif but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a Hif-independent fashion. Our results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.

SummaryGermline NF1, c-RET, SDH, and VHL mutations cause familial pheochromocytoma. Pheochromocytomas derive from sympathetic neuronal precursor cells. Many of these cells undergo c-Jun-dependent apoptosis during normal development as NGF becomes limiting. NF1 encodes a GAP for the NGF receptor TrkA, and NF1 mutations promote survival after NGF withdrawal. We found that pheochromocytoma-associated c-RET and VHL mutations lead to increased JunB, which blunts neuronal apoptosis after NGF withdrawal. We also found that the prolyl hydroxylase EglN3 acts downstream of c-Jun and is specifically required among the three EglN family members for apoptosis in this setting. Moreover, EglN3 proapoptotic activity requires SDH activity because EglN3 is feedback inhibited by succinate. These studies suggest that failure of developmental apoptosis plays a role in pheochromocytoma pathogenesis.

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-kappaB activity but the mechanism is unclear. NF-kappaB affects tumorigenesis and therapeutic resistance in some settings. We found that pVHL associates with the NF-kappaB 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-kappaB in VHL(+/+) cells, and Card9 siRNA normalized NF-kappaB 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 a ubiquitin conjugating enzyme and a kinase. The latter activity, which promotes Card9 phosphorylation, links pVHL to control of NF-kappaB activity and tumorigenesis.

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.