FAM96A is a novel pro-apoptotic tumor suppressor in gastrointestinal stromal tumors.

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.

Macromolecular complexes are essential players in numerous biological processes. They are often large, dynamic, and rather labile; approaches to study them are scarce. Covering masses up to ∼30 MDa, we separated the native complexome of rat heart mitochondria by blue-native and large-pore blue-native gel electrophoresis to analyze its constituents by mass spectrometry. Similarities in migration patterns allowed hierarchical clustering into interaction profiles representing a comprehensive analysis of soluble and membrane-bound complexes of an entire organelle. The power of this bottom-up approach was validated with well-characterized mitochondrial multiprotein complexes. TMEM126B was found to comigrate with known assembly factors of mitochondrial complex I, namely CIA30, Ecsit, and Acad9. We propose terming this complex mitochondrial complex I assembly (MCIA) complex. Furthermore, we demonstrate that TMEM126B is required for assembly of complex I. In summary, complexome profiling is a powerful and unbiased technique allowing the identification of previously overlooked components of large multiprotein complexes.

Dynamics of gut mucosal and systemic Th1/Th2 cytokine responses in interferon-gamma and interleukin-12p40 knock out mice during primary and challenge Cryptosporidium parvum infection.

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in the immunocompromised host. CD4+ T cells and interferon (IFN)-gamma play dominant roles in host immune response to infection. However, effector mechanisms that are responsible for recovery from infection are poorly understood. In the present study we analyzed mice deficient in IFN-gamma or interleukin (IL)-12 in parallel to C57BL/6 wild type mice as models for murine cryptosporidiosis. Our results identified IFN-gamma as the key cytokine in the innate as well as adaptive immunity during primary and also challenge C. parvum infection. Furthermore, both Th1 and Th2 cytokines appear to contribute to the resolution of a primary infection, the former being dominant over the latter. Dramatic changes in the expression of cytokine genes were seen in the ileum (the site of infection) but not in the mesenteric lymph nodes and spleen. During re-challenge, a significant increase of IFN-gamma was recorded in IL-12 deficient mice (IL-12KO). Additionally, we present data suggesting a contribution of IL-18 in resistance of C. parvum infection even in the absence of IFN-gamma. Anti-IL-18 antibody treatment led to increased susceptibility to infection in both strains of immunodeficient mice. Besides its function in inducing IFN-gamma in IL-12 knock out mice, IL-18 appears to be involved in the regulation of the Th1/Th2 responses in C. parvum. Neutralization resulted in a cytokine imbalance with up regulation of systemic (spleen) Th2 cytokine genes, notably IL-4 and IL-13. These data demonstrate that susceptibility or resistance to C. parvum infection depends on a delicate balance between the production of Th1 cytokines, needed to control parasite growth, and Th2 cytokines, to limit pathology.