The homeobox gene HLXB9 is upregulated in a morphological subset of poorly differentiated hepatocellular carcinoma

The prognostic outcome for hepatocellular carcinoma (HCC) remains poor. Disease progression is accompanied by dedifferentiation of the carcinoma, a process that is not well understood. The aim of this study was to get more insight into the molecular characteristics of dedifferentiated carcinomas using high throughput techniques. Microarray-based global gene expression analysis was performed on five poorly differentiated HCC cell lines compared with non-neoplastic hepatic controls and a set of three cholangiolar carcinoma (CC) cell lines. The gene with the highest upregulation was HLXB9. HLXB9 is a gene of the homeobox genfamily important for the development of the pancreas. RT-PCR confirmed the upregulation of HLXB9 in surgical specimens of carcinoma tissue, suggesting its biological significance. Interestingly, HLXB9 upregulation was primary observed in poorly differentiated HCC with a pseudoglandular pattern compared with a solid pattern HCC or in moderate or well-differentiated HCC. Additional the expression of translated HLXB9, the protein HB9 (NCBI: NP_001158727), was analyzed by western blotting. Expression of HB9 was only detected in the cytoplasm but not in the nuclei of the HCC cells. For validation CC were also investigated. Again, we found an upregulation of HLXB9 in CC cells accompanied by an expression of HB9 in the cytoplasms of these tumor cells, respectively. In conclusion, homeobox HLXB9 is upregulated in poorly differentiated HCC with a pseudoglandular pattern. The translated HB9 protein is found in the cytoplasm of these HCC and CC. We therefore assume HLXB9 as a possible link in the understanding of the development of HCC and CC, respectively.

Hormonally defined pancreatic and duodenal neuroendocrine tumors differ in their transcription factor signatures: expression of ISL1, PDX1, NGN3, and CDX2

We recently identified the transcription factor (TF) islet 1 gene product (ISL1) as a marker for well-differentiated pancreatic neuroendocrine tumors (P-NETs). In order to better understand the expression of the four TFs, ISL1, pancreatico-duodenal homeobox 1 gene product (PDX1), neurogenin 3 gene product (NGN3), and CDX-2 homeobox gene product (CDX2), that mainly govern the development and differentiation of the pancreas and duodenum, we studied their expression in hormonally defined P-NETs and duodenal (D-) NETs. Thirty-six P-NETs and 14 D-NETs were immunostained with antibodies against the four pancreatic hormones, gastrin, serotonin, calcitonin, ISL1, PDX1, NGN3, and CDX2. The TF expression pattern of each case was correlated with the tumor's hormonal profile. Insulin-positive NETs expressed only ISL1 (10/10) and PDX1 (9/10). Glucagon-positive tumors expressed ISL1 (7/7) and were almost negative for the other TFs. Gastrin-positive NETs, whether of duodenal or pancreatic origin, frequently expressed PDX1 (17/18), ISL1 (14/18), and NGN3 (14/18). CDX2 was mainly found in the gastrin-positive P-NETs (5/8) and rarely in the D-NETs (1/10). Somatostatin-positive NETs, whether duodenal or pancreatic in origin, expressed ISL1 (9/9), PDX1 (3/9), and NGN3 (3/9). The remaining tumors showed labeling for ISL1 in addition to NGN3. There was no association between a particular TF pattern and NET features such as grade, size, location, presence of metastases, and functional activity. We conclude from our data that there is a correlation between TF expression patterns and certain hormonally defined P-NET and D-NET types, suggesting that most of the tumor types originate from embryologically determined precursor cells. The observed TF signatures do not allow us to distinguish P-NETs from D-NETs.

Consistent nuclear expression of thyroid transcription factor 1 in subependymal giant cell astrocytomas suggests lineage-restricted histogenesis.

Thyroid transcription factor 1 (TTF-1) is encoded by the NKX2-1 homeobox gene. Besides specifying thyroid and pulmonary organogenesis, it is also temporarily expressed during embryonic development of the ventral forebrain. We recently observed widespread immunoreactivity for TTF-1 in a case of subependymal giant cell astrocytoma (SEGA, WHO grade I) – a defining lesion of the tuberous sclerosis complex (TSC). This prompted us to investigate additional SEGAs in this regard. We found tumor cells in all 7 specimens analyzed to be TTF-1 positive. In contrast, we did not find TTF-1 immunoreactivity in a cortical tuber or two renal angiomyolipomas resected from TSC patients. We propose our finding of consistent TTF-1 expression in SEGAs to indicate lineage-committed derivation of these tumors from a regionally specified cell of origin. The medial ganglionic eminence, ventral septal region, and preoptic area of the developing brain may represent candidates for the origin of SEGAs. Such lineagerestricted histogenesis may also explain the stereotypic distribution of SEGAs along the caudate nucleus in the lateral ventricles.

The bovine aristaless-like homeobox 4 (ALX4) as a candidate gene for syndactyly

The ALX4 (aristaless-like homeobox 4) gene encodes a paired-type homeodomain transcriptional activator and plays a major role in anterior-posterior pattern formation during limb development. Here, the cloning, genomic structure and expression of the bovine ortholog of the ALX4 gene are reported. The bovine ALX4 gene consists of four exons and is located on BTA15q28-->q29 in a region syntenic to HSA11p11.2. The transcribed ALX4 mRNA encodes a 397-amino-acid protein showing a paired-type homeodomain and a C-terminal stretch of amino acids known as the OAR- or aristaless domain. The predicted protein shares 92.5% identity to human and mouse ALX4 proteins and all three species share almost complete identity in the conserved domains. ALX4 expression was detected by reverse transcriptase polymerase chain reaction in bovine fetal limb bones. The ALX4 gene was evaluated as a candidate gene for bovine syndactyly which has been mapped on the telomeric region of cattle chromosome 15. Sequencing of the four exons with flanking sequences of the bovine ALX4 gene from a panel of 14 affected animals belonging to German Holstein, German Fleckvieh and crossbreds, and 27 unaffected individuals from German Holstein revealed five silent SNPs within the coding region out of eleven SNPs in total. Four SNPs were polymorphic in the affected animals, but in comparison to the genotyped unaffected individuals the genotype distribution showed no evidence for an association to the phenotype. Therefore our data indicate that the ALX4 gene can probably be excluded as candidate gene for bovine syndactyly in the examined animals.

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.