Cancer-associated fibroblasts (CAFs) provide critical metabolites for tumor growth and undergo metabolic reprogramming to support glycolysis. However, the molecular mechanisms responsible for this change remain unclear. Here, we report that TGF-β1- or PDGF-induced CAFs switch from oxidative phosphorylation to aerobic glycolysis. We identify downregulation of isocitrate dehydrogenase 3α (IDH3α) as a marker for this switch. Furthermore, miR-424 downregulates IDH3α during CAF formation. Downregulation of IDH3α decreases the effective level of α-ketoglutarate (α-KG) by reducing the ratio of α-KG to fumarate and succinate, resulting in PHD2 inhibition and HIF-1α protein stabilization. The accumulation of HIF-1α, in turn, promotes glycolysis by increasing the uptake of glucose, upregulating expression of glycolytic enzymes under normoxic conditions, and inhibiting oxidative phosphorylation by upregulating NDUFA4L2. CAFs from tumor samples exhibit low levels of IDH3α, and overexpression of IDH3α prevents transformation of fibroblasts into CAFs. Our studies reveal IDH3α to be a critical metabolic switch in CAFs.

Saturable and reverse saturable absorption at longitudinal surface plasmon resonance (SPR) in gold nanorods (Au NRs) have been observed using Z-scan and transient absorption techniques with femtosecond laser pulses. At lower excitation irradiances, the wavelength dispersion of saturable absorption has been determined near the longitudinal mode of SPR with a recovery time determined to be a few ten picoseconds on the SPR resonance. With higher excitation irradiances, reverse saturable absorption occurs and becomes dominant. The underlying mechanisms are discussed.Such reversible saturable absorption makes Au NRs an ideal candidate for optical limiting applications.

Abstract:We report a systematic investigation of both three-photon absorption (3PA) spectra and wavelength dispersions of Kerr-type nonlinear refraction in wide-gap semiconductors. The Z-scan measurements are recorded for both ZnO and ZnS with femtosecond laser pulses. While the wavelength dispersions of the Kerr nonlinearity are in agreement with a two-band model, the wavelength dependences of the 3PA are found to be given by (3E photon /E g -1)5/2 (3E photon /E g ) -9 . We also evaluate higher-order nonlinear optical effects including the fifth-order instantaneous nonlinear refraction associated with virtual three-photon transitions, and effectively seventh-order nonlinear processes induced by three-photon-excited free charge carriers. These higher-order nonlinear effects are insignificant with laser excitation irradiances up to 40 GW/cm 2 . Both pump-probe measurements and three-photon figures of merits demonstrate that ZnO and ZnS should be a promising candidate for optical switching applications at telecommunication wavelengths. 1951-1955 (1997

How TGF-β1-mediated signaling pathways are finely tuned to orchestrate the generation of carcinoma-associated fibroblasts (CAFs) is poorly understood. Here, we demonstrate that miR-21 and the signaling of its target Smad 7 determine TGF-β1-induced CAF formation. In primary cultured fibroblasts, mature miR-21 increases after TGF-β1 treatment, whereas the Smad 7 protein level decreases. MiR-21 binds to the 3′ UTR of Smad7 mRNA and inhibits its translation, rather than causing its degradation. Most importantly, Smad 7 is bound to Smad 2 and 3, which are thought to competitively bind to TGFBR1, and prevents their activation upon TGF-β1 stimulation. The depletion of miR-21 or the overexpression of Smad 7 blocks TGF-β1-induced CAF formation, whereas the overexpression of miR-21 or the depletion of Smad 7 promotes CAF formation, even without TGF-β1 stimulation. Collectively, these findings clearly demonstrate that miR-21 and Smad7 are critical regulators of TGF-β1 signaling during the induction of CAF formation.

CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin ␣1, ␣3, and ␣5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore, the nuclear localization of CUL4B mediated by NLS is critical for its normal function in cell proliferation.Cullins function as a "scaffold" in cullin-RING-based E3 ubiquitin ligases (CRLs).3 CRLs constitute a major subclass of RING finger E3s that regulate diverse cellular processes, including cell cycle progression, transcription, signal transduction, and development (1, 2). Cullins are evolutionarily conserved from yeast to mammals. Although sequence homology spans the entire protein, the C terminus, characterized by the ϳ200-amino acid cullin homology domain, is most conserved. Humans encode seven cullin members, CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7 (2). CUL4A and CUL4B are derived from one ancestor, CUL4, which exists in Schizosaccharomyces pombe (Pcu4), Xenopus laevis, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana but is absent in Saccharomyces cerevisiae. CUL4A and CUL4B also exist in other higher organisms, including zebrafish and the mouse (3). The protein sequences between human CUL4A and CUL4B are 83% identical, with CUL4B having a unique N terminus of 149 amino acids (supplemental Fig. S1). CUL4A CRL complexes were shown to contain Rbx1 and the adaptor protein DDB1. DDB1 interacts with WD-40 repeat motif-containing proteins that determine the substrate specificity of the CUL4A ubiquitin ligase complex (3-7). Loss of Cul4 in Drosophila cells leads to G 1 arrest that is associated with an increase in the cyclin-dependent kinases (CDK) inhibitor Dacapo (8). Recent genetic studies have shown that mutations in CUL4B gene can cause an X-linked mental retardation syndrome (9, 10). However, no germ line mutation in human CUL4A has been reported, although overexpression of ...

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1γ1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

Tumor relapse after radiotherapy is a significant challenge to oncologists, even after recent the advances in technologies. Here, we showed that cancer-associated fibroblasts (CAFs), a major component of cancer stromal cells, promoted irradiated cancer cell recovery and tumor relapse after radiotherapy. We provided evidence that CAFs-produced IGF1/2, CXCL12 and β-hydroxybutyrate were capable of inducing autophagy in cancer cells post-radiation and promoting cancer cell recovery from radiation-induced damage in vitro and in vivo in mice. These CAF-derived molecules increased the level of reactive oxygen species (ROS) post-radiation, which enhanced PP2A activity, repressing mTOR activation and increasing autophagy in cancer cells. Consistently, the IGF2 neutralizing antibody and the autophagy inhibitor 3-MA reduce the CAF-promoted tumor relapse in mice after radiotherapy. Taken together, our findings demonstrated that CAFs promoted irradiated cancer cell recovery and tumor regrowth post-radiation, suggesting that targeting the autophagy pathway in tumor cells may be a promising therapeutic strategy for radiotherapy sensitization.

ATM is a central mediator of the cellular response to the DNA damage produced by ionizing radiation. We recently showed that protein phosphatase 1 (PP1) is activated by ATM. Because Nek2 is activated by autophosphorylation, and because its dephosphorylation is catalyzed by PP1, we asked if the radiation damage signal to Nek2 was mediated by PP1. Overexpression of Nek2 induces premature centrosome splitting probably by phosphorylating centrosome cohesion proteins C-Nap1 and Rootletin. In this study, we show isoform specificity of PP1 binding and regulation of Nek2. Although both PP1A and PP1; coimmunoprecipitated with Nek2, only PP1A regulated Nek2 function. Ionizing radiation inhibited Nek2 activity, and this response was dependent on ATM and on PP1 binding to Nek2 and coincident with Thr 320 dephosphorylation of PP1. Radiation-induced inhibition of centrosome splitting was abrogated in cells expressing Nek2 mutated in the PP1-binding motif outside the kinase domain. Conversely, cells depleted of PP1A by small interfering RNA showed enhanced centrosome splitting and loss of radiationinduced inhibition of centrosome splitting. The identification of a PP1-specific isoform mediating a checkpoint response opens up the possibility of selectively targeting phosphatases as novel radiation sensitizers. [Cancer Res 2007;67(3):1082-9]