Peutz-Jeghers (PJ) syndrome is an autosomal-dominant disorder characterized by melanocytic macules of the lips, multiple gastrointestinal hamartomatous polyps and an increased risk for various neoplasms, including gastrointestinal cancer. The PJ gene was recently mapped to chromosome 19p13.3 by linkage analysis, with the highest lod score at marker D19S886. In a distance of 190 kb proximal to D19S886, we identified and characterized a novel human gene encoding the serine threonine kinase STK11. In a three-generation PJ family, we found an STK11 allele with a deletion of exons 4 and 5 and an inversion of exons 6 and 7 segregating with the disease. Sequence analysis of STK11 exons in four unrelated PJ patients has identified three nonsense and one acceptor splice site mutations. All five germline mutations are predicted to disrupt the function of the kinase domain. We conclude that germline mutations in STK11, probably in conjunction with acquired genetic defects of the second allele in somatic cells, cause the manifestations of PJ syndrome.

Hypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis. It represents an attractive therapeutic target in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis. In HIF-1alpha knockdown DLD-1 colon cancer cells (DLD-1(HIF-kd)), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1alpha (DLD-1(HIF-wt)). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1(HIF-kd) but not DLD-1(HIF-wt) cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-kappaB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1(HIF-kd) but not DLD-1(HIF-wt) xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1alpha may be most effective when IL-8 is simultaneously targeted.

A BSTR ACTVasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP-and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)͞VASP protein family and is thought to be involved in actin filament formation and integrin ␣ IIb ␤ 3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP؊͞؊ mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin ␣ IIb ␤ 3 activation. Although the limited phenotypic differences between wild-type and VASP؊͞؊ mice suggested functional compensation of VASP by members of the Ena͞VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.Vasodilator-stimulated phosphoprotein (VASP) initially was discovered and characterized as a prominent substrate for both cGMP-dependent protein kinases (cGKs) and cAMP-dependent protein kinases (cAKs) in human platelets (1, 2). VASP is phosphorylated in vitro and in intact human platelets at serine-157, serine-239, and threonine-278 by both cAK and cGK, and it is in vitro and in intact human platelet dephosphorylated by protein phosphatases I and II with overlapping selectivity (3-7). Phosphorylation of serine-157, the site preferred by cAK, leads to a shift in the apparent molecular mass of VASP in SDS͞PAGE from 46 kDa to 50 kDa (5, 6). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e., cAMP-elevating prostaglandins and cGMP-elevating NO donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the human platelet integrin ␣ IIb ␤ 3 (3,8).

Cells transiently adapt to hypoxia by globally decreasing protein translation. However, specific proteins needed to respond to hypoxia evade this translational repression. The mechanisms of this phenomenon remain unclear. We screened for and identified small molecules that selectively decrease HIF-2a translation in an mTOR independent manner, by enhancing the binding of Iron Regulatory Protein 1 (IRP1) to a recently reported Iron-Responsive Element (IRE) within the 5’-untranslated region (UTR) of the HIF-2a message. Knocking down the expression of IRP1 by shRNA abolished the effect of the compounds. Hypoxia de-represses HIF-2a translation by disrupting the IRP1- HIF-2a IRE interaction. Thus, this chemical genetic analysis describes a molecular mechanism by which translation of the HIF-2a message is maintained during conditions of cellular hypoxia through inhibition of IRP-1 dependent repression. It also provides the chemical tools for studying this phenomenon.

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The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis, and the hypoxia-inducible factor-1 (HIF-1) transcription factor is known to be a key mediator of this process. In colon cancer, the frequently mutated K-ras oncogene also can regulate VEGF expression, but the role that K-ras may play in hypoxia is unknown. Hypoxia induced VEGF promoter activity, mRNA, and protein levels in colon cancer cells. Although HIF-1␣ was induced by hypoxia, VEGF reporter constructs with selectively mutated hypoxia-response elements remained responsive to hypoxia. In addition, "knockdown" of HIF-1␣ by RNA interference only minimally inhibited the hypoxic induction of VEGF. A region of the VEGF promoter between ؊420 and ؊90 bp mediated this HIF-independent induction by hypoxia. The introduction of K-ras Val12 augmented the hypoxic induction of VEGF, and this was observed in wild-type and HIF-1␣ knockdown colon cancer cells. Thus, VEGF may be induced by hypoxia through HIF-dependent and HIFindependent pathways, and K-ras also can induce VEGF in hypoxia independent of HIF-1. These findings suggest the existence of multiple mechanisms regulating the hypoxic induction of VEGF in colon cancer.