In Caenorhabditis elegans, lin-2, lin-7, and lin-10 genetically interact to control the trafficking of the Let-23 growth factor receptor to the basolateral surface of body epithelia. The human homologue of the lin-10 gene has recently been identified as a member of the X11 gene family. The X11 proteins contain one phosphotyrosine binding (PTB) and two PSD-95⅐Dlg⅐ZO-1 (PDZ) domains as well as an extended amino terminus. We have previously shown that the PTB domain of X11␣ (also known as Mint1) can bind to the amyloid precursor protein (APP) in a phosphotyrosine-independent fashion and can markedly inhibit the processing of APP to the amyloid ␤ (A␤) peptide. Here, we report that X11␣ directly binds to the mammalian homologue of Lin-2 (mLin-2), also known as CASK. This binding is mediated by direct interaction between the Calmodulin Kinase II (CKII)-like domain of mLin-2 and the amino terminus of X11␣. Furthermore, we can detect direct interactions between mLin-2 and mammalian Lin-7 (mLin-7). In mouse brain, we have identified a heterotrimeric complex that contains mLin-2, mLin-7, and X11␣ and that is likely important for the localization of proteins in polarized cells. This complex may play an important role in the trafficking and processing of APP in neurons.

A natural BH3-mimetic, small molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing Phase II-III clinical trials for human prostate cancer. Here we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy via blocking Bcl-2—Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1 and activating the autophagic pathway. (-)-Gossypol-induced autophagy is Beclin1- and Atg5-dependent. Our results demonstrate for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which would facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.The proper targeting of proteins at the apical or basolateral surface of epithelial cells is crucial for cellular transport. Similarly, proper targeting of proteins to either axons or dendrites plays an important role in neurotransmission. Many studies have been directed at the molecular basis that controls these targeting decisions. In Caenorhabditis elegans, three proteins (Lin-2, Lin-7, and Lin-10) are crucial for the correct localization of the worm epidermal growth factor receptor, Let-23, to the basolateral surface of the body wall epithelium (1-3). The lin-10 gene has also been shown to have a role in localization of glutamate receptors in the C. elegans brain (4). A similar complex of mammalian homologues of Lin-2, Lin-7, and Lin-10 have been identified in mammalian brain (5, 6). In mammalian brain, the homologue of Lin-10 is known as X11␣ or Mint1 (7-9). X11␣/Mint1 contains one PTB 1 domain and two PDZ domains. PTB domains act as a phosphotyrosine binding domain in proteins such as Shc and insulin receptor substrate-1, but, in X11 and many other proteins, the PTB domain binding targets bind independent of phosphotyrosine (10). PDZ domain proteins are named for their first identification in PSD-95, Drosophila Discs Large, and zona occludens-1 proteins (11). They commonly bind to sequences at the extreme carboxyl terminus of proteins. In addition to PTB and PDZ domains, X11␣/Mint1 contains an extended amino terminus that can bind mLin-2/Cask and Munc-18 (9, 12).mLin-2/CASK is a membrane-associated guanylate kinase (Maguk) protein containing a guanylate kinase-like domain, an SH3 domain, a PDZ domain, and a calmodulin kinase-like domain (1...

SUMMARY FBXW7 is a haploinsufficient tumor suppressor with loss-of-function mutations occurring in human cancers. FBXW7 inactivation causes genomic instability, yet the mechanism remains elusive. Here we show that FBXW7 facilitates non-homologous end-joining (NHEJ) repair and FBXW7 depletion causes radiosensitization. In response to ionizing radiation, ATM phosphorylates FBXW7 at serine 26 to recruit it to DNA double-strand break (DSB) sites, while activated DNA-PKcs phosphorylates XRCC4 at serines 325/326 which promotes binding of XRCC4 to FBXW7. SCFFBXW7 E3 ligase then promotes polyubiquitylation of XRCC4 at lysine 296 via K63-linkage for enhanced association with the Ku70/80 complex to facilitate NHEJ repair. Consistent with these findings, a small molecule inhibitor that abrogates XRCC4 polyubiquitylation reduces NHEJ repair. Our study demonstrates one mechanism by which FBXW7 contributes to genome integrity and implies that inactivated FBXW7 in human cancers could be a strategy for increasing efficacy of radiotherapy.

Purpose While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 [poly (ADP-ribose) polymerase 1/2] inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. Experimental Design Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX) and HRR (homologous recombination repair) were determined. Results Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization while treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, while AZD1775 produced moderate, yet significant, radiosensitization (P<0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P<0.0001) evidenced by a 13-day delay in tumor volume doubling (vs radiation alone) and complete eradication of 20% of tumors. Conclusions Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization.