Apoptosis, as a special form of programmed cell death (PCD) is an essential developmental process that is involved in tissue homeostasis and, whose deregulation has severe pathological implications for an organism. Thus, the evasion of the apoptotic program is one hallmark capability of cancer cells. Tumor suppressor genes (TSG) under normal conditions fulfill functions like sensing DNA-damage or regulating gene expression and they may contribute to the induction of apoptosis. In most cancer cells, TSG are mutated, inactivated or their expression level is reduced to enable neoplastic outgrowth and tumor cell proliferation. Furthermore, inactivation or blockage of TSG function may contribute to activation and upregulation of anti-apoptotic oncogenes. The identification and functional characterization of novel pro-apoptotic proteins, which can act as tumor suppressors themselves, is important in cancer research.
In this study, the novel APAF-1 binding protein FAM96A, which was discovered in a yeast two hybrid screen, was functionally characterized, and its involvement in tumorigenesis and Fe-S cluster biogenesis was elucidated. Importantly, the endogenous interaction with monomeric APAF-1 was confirmed.
Furthermore, FAM96A has recently been described to interact with the cytosolic iron sulfur assembly protein Ciao1. A combined functional contribution of Ciao1 and FAM96A to intrinsic apoptosis was excluded by overexpression and knockdown experiments during this study. Ciao1 exhibits a rather anti-apoptotic function, which is independent of FAM96A and, most probably, connected to its role in cytosolic Fe-S biogenesis. However, a scaffolding function of FAM96A during cytosolic iron-sulfur assembly might reflect a conserved function of the protein.
Ciao1 was described to regulate the transcriptional activity of WT1, a tumor suppressor in childhood nephroblastoma. Here, a reduction of WT1 mRNA expression upon simultaneous Ciao1 overexpression was observed, whereas the contribution of FAM96A to the regulation of the WT1 transcription factor was only minor.
The role of FAM96A in intrinsic apoptosis was investigated by stable lentiviral protein overexpression in cancer cells. Human colon carcinoma and murine renal cancer cells were transduced to upregulate their FAM96A levels. These stable cell lines were compared to control vector transduced cells in apoptosis assays investigating the effects of FAM96A overexpression on the intrinsic pathway. Upon induction of PCD by the chemotherapeutic agents mitomycin C and the second-generation platinum derivative oxaliplatin, cell killing was enhanced when FAM96A was simultaneously upregulated. Accordingly, when protein levels were diminished as a consequence of lentiviral short hairpin RNA (shRNA)-mediated knockdown, a significant desensitization of murine fibroblast and RKO cells to the induction of the intrinsic apoptosis pathway was observed. Furthermore, the influence of increased FAM96A expression levels was investigated in vivo in subcutaneous tumor xenograft experiments in NOD/SCID mice. The engraftment potential of colon cancer cells was drastically impaired in FAM96A groups compared to controls, and tumor growth was reduced in RKO as well as Renca-lacZ tumors. While a regulatory influence of FAM96A on intrinsic apoptosis could be observed, the cell cycle distribution of Renca-lacZ was not affected by FAM96A upregulation.
Although several approaches were taken to elucidate the molecular mechanism of the pro-apoptotic contribution of FAM96A to intrinsic apoptosis and its putative function during apoptosome assembly, it remains unresolved until now. A potential scaffolding function of FAM96A during apoptosome assembly or an involvement as a nucleotide exchange factor on APAF-1 cannot be excluded. As a pro-apoptotic protein, FAM96A resembles a putative tumor-suppressor gene, and losses within the FAM96A locus were detected by comparative genomic hybridization. Here, copy number aberrations were observed most prominently in gastrointestinal stromal tumors (GIST) where 48.8% of tumors analyzed indicated deletions. This was further addressed by immunohistochemistry of 53 GIST patient samples, and independent of cancer origin, no positive staining for FAM96A could be observed. A second patient cohort of GISTs of distinct grading was analyzed for FAM96A mRNA expression, which was reduced in 29 out of 31 samples. Upon re-introduction of FAM96A in established GIST cell lines, susceptibility towards apoptotic treatment with the state-of-the-art tyrosine kinase inhibitor Imatinib Mesylate and, in the case of imatinib-resistant GIST, staurosporine was increased compared to controls. Tumorigenicity of GIST882 cells re-expressing FAM96A was significantly reduced in a tumor xenograft experiment, and the engraftment potential was diminished. Quantitative real time PCR analysis on the time point of FAM96A loss in the course of tumorigenesis indicate that reduction of FAM96A mRNA levels may occur as an oncogenic event during malignant transformation of interstitial cells of Cajal, the suspected cell of origin of GIST. FAM96A therefore represents a marker for GIST and a novel tumor suppressor protein in this cancer entity. Nonetheless, FAM96A exhibits its tumor-suppressive potential not only in GIST, but also in further tumor entities as suggested by multi-tumor tissue arrays. Protein levels were reduced in renal and transitional cell carcinoma compared to normal kidney and bladder tissue.
The physiological function of the protein was further investigated by the establishment and preliminary analysis of a conditional FAM96A knockout mouse model. Here, FAM96A/lacZ reporter mice were generated in which Exon 3 of the FAM96A gene was deleted following breeding of mice with CMV-Cre mice. Strong endogenous FAM96A promoter activity was observed in tissues of the immune system such as spleen and thymus, and in the gastrointestinal tract of the FAM96A-/- mice. Surprisingly, and in contrast to observations from yeast, drosophila and zebrafish model systems, where knockout of the gene was lethal, FAM96A-/- mice were born and vital and displayed no obvious phenotype under non-stressed conditions
