Point mutations that activate the Ki-ras proto-oncogene are presented in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-myc. Thus, the activated Ki-ras gene plays a key role in colorectal tumorigenesis through altered cell differentiation and cell growth.

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression. ( IntroductionCancer is believed to arise and progress toward increasing malignancy as a result of cumulative genetic "hits" sustained by the tumor cell genome. Paradigmatic in this regard is the development of colorectal carcinoma (CRC), where sequential transition through clinical stages of the disease is paralleled by a series of well-characterized alterations in proto-oncogenes and tumor suppressor genes. 1 In this tumor type, activation of mutant K-ras and subsequent inactivation/loss of p53 are key changes, which drive many interrelated aspects of the malignant phenotype including aberrant mitogenesis and survival. 2 Moreover, both of these genetic alterations are thought to contribute to proangiogenic properties of affected cancer cells, 3,4 and thereby enable them to exploit the host vascular system to advance malignant growth and metastasize in vivo. 5 The involvement of the vascular system in malignancy encompasses not only angiogenesis but also systemic hypercoagulability. Blood clotting abnormalities are detected in up to 90% of patients with metastatic disease, and thrombosis represents the second most frequent cause of cancer-related mortality. 6 Cancer coagulopathy is often linked to up-regulation of tissue factor (TF), the primary cellular initiator of the blood coagulation cascade. 7,8 Interaction of coagulation factor VIIa with TF on the cell surface leads to activation of factor X and generation of thrombin, with subsequent involvement of platelets and formation of a fibrin clot. 9 Remarkably, as a member of the class II cytokine receptor family, TF is also capable of transducing intracellular signals and regulating gene expression. 10,11 Interestingly, elements of the coagulation/fibrinolytic system in general, 12 and TF in particular, have been implicated in regulation of angiogenesis, 13,14 as well as tumor growth 15 and metastasis 16 in various experimental settings. This is consistent with the observed up-regulation of TF in huma...

SUMMARY PIK3CA and PTEN alterations are common in human cancer, but only a fraction of such tumors are dependent upon AKT signaling. AKT-independence is associated with redundant activation of cap-dependent translation mediated by convergent regulation of the translational repressor 4E-BP1 by the AKT and ERK pathways. This provides mechanistic bases for the limited activity of AKT and MEK inhibitors in tumors with co-mutation of both pathways and the profound synergy observed with combined inhibition. Whereas such tumors are sensitive to a dominant active 4E-BP1 mutant, knockdown of 4E-BP1 expression reduces their dependence on AKT/ERK signaling for translation or survival. Thus, 4E-BP1 plays a prominent role in mediating the effects of these pathways in tumors in which they are activated by mutation.

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Rheumatoid arthritis (RA) is a common autoimmune disease with a complex genetic etiology. Herein we identify a single-nucleotide polymorphism (SNP) in the promoter region of FcRH3, a member of the Fc receptor homolog family, that is associated with RA susceptibility (OR=2. 15, P=0.00000085). This polymorphism alters the binding affinity of nuclear factor-κB and regulates NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptFcRH3 expression. High FcRH3 expression on B-cells and augmented autoantibody production were observed in individuals with the disease-susceptible genotype. Associations were also found between the SNP and susceptibility to autoimmune thyroid disease and systemic lupus erythematosus. FcRH3 may thus play a pivotal role in autoimmunity.Rheumatoid arthritis represents one of the most common autoimmune diseases, and is characterized by inflammation of synovial tissue and joint destruction. Although the disease is believed to result from a combination of genetic and environmental factors, the complete etiology of RA has not yet been clarified 1 . While specific haplotypes of human leukocyte antigen (HLA)-DRB1, usually referred to as shared-epitope (SE) sequences 2 , have been repeatedly reported as conferring RA-susceptibility 3,4 , other genetic components are also involved in the pathogenesis of RA 5 . This combination of HLA haplotypes and non-HLA genes accounting for disease susceptibility is also seen in other autoimmune diseases 6-8 . In autoimmune thyroid disease (AITD), for instance, studies have consistently shown that the HLA-DR3 haplotype is associated with disease risk, in addition to a functional haplotype of a non-HLA gene, CTLA4, that has recently been associated with AITD susceptibility 9 .Identification of non-HLA genes associated with RA susceptibility and other autoimmunities seems difficult, due to the low relative risk of disease resulting from these non-HLA genes compared with the strong relative risk from disease-associated HLA haplotypes. In a search for non-HLA determinants of disease susceptibility, whole genome studies have been conducted for both human autoimmune diseases and experimental animal models. These studies have revealed non-random clustering of susceptibility loci for clinically distinct diseases 8,10 . This overlapping of susceptibility loci for multiple autoimmunities suggests the existence of common susceptibility genes in those regions. Intense studies of loci-clustering regions has revealed genes commonly associated with multiple autoimmune diseases, such as CTLA4 on 2q33 (ref. and Idd17 (ref. 25)). Although 1q21-23 is a strong candidate region for RA susceptible genes, as above mentioned, the association of classical FcγRs with disease susceptibility remains controvertial 26,27 . The present study focused on the 1q21-23 region to identify RA-associated genes in Japanese subjects using linkage disequilibrium (LD) mapping. RESULTS Case-control study by SNP-based LD-mapping in 1q21-23To evaluate the extent of association, we a...

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing ("winner") and engulfed ("loser") cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors.

Most neurons in vertebrates make a developmental choice between two principal neurotransmitter phenotypes (glutamatergic versus GABAergic). Here we show that the homeobox gene Lbx1 determines a GABAergic cell fate in the dorsal spinal cord at early embryonic stages. In Lbx1-/- mice, the presumptive GABAergic neurons are transformed into glutamatergic cells. Furthermore, overexpression of Lbx1 in the chick spinal cord is sufficient to induce GABAergic differentiation. Paradoxically, Lbx1 is also expressed in glutamatergic neurons. We previously reported that the homeobox genes Tlx1 and Tlx3 determine glutamatergic cell fate. Here we show that impaired glutamatergic differentiation, observed in Tlx3-/- mice, is restored in Tlx3-/-Lbx1-/- mice. These genetic studies suggest that Lbx1 expression defines a basal GABAergic differentiation state, and Tlx3 acts to antagonize Lbx1 to promote glutamatergic differentiation.