CUX1: target of Akt signalling and mediator of resistance to apoptosis in pancreatic cancer.

BACKGROUND AND AIMS: The transcription factor CUX1 is known as a regulator of cell differentiation and cell cycle progression. Previously, CUX1 was identified as a modulator of invasiveness in various cancers. Based on expression profiles suggesting a role for CUX1 in mediating chemoresistance, the aim of this study was to characterise the effect of CUX1 on apoptosis as well as its regulation by signalling pathways modulating drug resistance in pancreatic cancer. METHODS: The effect of CUX1 on TRAIL- (tumour necrosis factor-related apoptosis-inducing ligand) and drug-induced apoptosis was analysed using overexpression and knock-down strategies. Regulation of CUX1 by phosphatidylinositol-3-kinase (PI3K)/Akt signalling was examined at the mRNA and protein level. The effect of CUX1 knock-down by nanoparticle-complexed small interfering RNA (siRNA) in vivo was analysed in a murine xenograft model. Furthermore, CUX1 RNA and protein expression was evaluated in human pancreatic cancer and adjacent normal tissues. RESULTS: Knock-down of CUX1 resulted in significantly enhanced TRAIL- and drug-induced apoptosis, associated with increased PARP (poly ADP-ribose polymerase) cleavage and caspase activity. Vice versa, overexpression of CUX1 inhibited apoptosis. CUX1 expression was induced by activation of Akt/protein kinase B signalling, and decreased by PI3K inhibitors. The antiapoptotic effect of CUX1 was associated with upregulation of BCL2 and downregulation of tumour necrosis factor alpha. CUX1 was significantly overexpressed in pancreatic cancers, as analysed by in situ hybridisation and immunohistochemistry. In vivo, silencing of CUX1 by intratumourally administered polyethylenimine-complexed siRNA led to reduced tumour growth and increased apoptosis in pancreatic cancer xenografts. CONCLUSION: CUX1 was identified as an important mediator of tumour cell survival in pancreatic cancer in vitro and in vivo.

WNT5A--target of CUTL1 and potent modulator of tumor cell migration and invasion in pancreatic cancer.

Previously, we have identified the transcription factor CUTL1 as an important mediator of tumor invasion and target of tumor growth factor-beta. Using high-throughput approaches, we identified several putative downstream effectors of CUTL1, among them WNT5A, a secreted member of the Wnt multigene family. The aim of this study was to investigate the role of WNT5A as a novel target of CUTL1 in pancreatic cancer. CUTL1 and WNT5A were stably over-expressed as well as transiently and stably knocked down by RNA interference. Effects on proliferation, migration and invasiveness were investigated by thymidine incorporation, Boyden chamber experiments and time-lapse microscopy. Expression of WNT5A in pancreatic cancer tissues was analyzed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. We found that CUTL1 transcriptionally up-regulated WNT5A on RNA, protein and promoter level. WNT5A significantly enhanced migration, proliferation and invasiveness, mediating the pro-invasive effects of CUTL1 to a major extent. WNT5A effects were accompanied by a marked modulation of marker genes associated with epithelial-mesenchymal transition. Using RT-PCR and immunohistochemistry, we found that WNT5A is up-regulated early during pancreatic cancerogenesis in pancreatic intraepithelial neoplasias lesions and in invasive pancreatic adenocarcinomas, as compared with normal pancreas tissues. These data identify WNT5A as important target of CUTL1 and as novel mediator of invasiveness and tumor progression in pancreatic cancer.

The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases.

6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P. patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190,731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'- and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Saccharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS- and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. patulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.

Isolation and sequence analysis of the fatty acid synthetase FAS2 gene from Penicillium patulum.

The fatty acid synthetase complex of Penicillium patulum was isolated and shown to be structurally similar to other known fungal fatty acid synthetases. It is composed of two subunit, alpha and beta, each with a molecular mass of about 200 kDa. P. patulum genomic and cDNA libraries were constructed in lambda gt11 and EMBL3 vectors. From these libraries, the P. patulum FAS2 gene together with its flanking DNA was isolated. The cloned genomic DNA was sequenced over a length of 6357 base pairs. The coding sequence of fatty acid synthetase subunit alpha, being 5571 nucleotides long, was identified within this DNA segment. The FAS2 gene is a mosaic of three exons (514, 4949 and 108 base pairs) and two introns, each of 54 base pairs in length. Both introns were absent in the corresponding cDNA sequences. Like other fungal introns both contain an internal CTAAC sequence, located 10 base pairs upstream of their 3'-exon/intron boundaries. In addition, they have, at their ends, the GTCAAGT and TAG consensus sequences characteristic of all eucaryotic introns. Furthermore, two pairs of direct repeats, of as yet unknown significance, were found in the two P. patulum introns. The P. patulum FAS2 gene encodes a protein of 1857 amino acids and 204.5 kDa molecular mass. It is 90 nucleotides shorter than the corresponding S. cerevisiae gene. In both organisms, the FAS2 genes and their products exhibit a high degree of overall sequence similarity at both the DNA (63%) and protein (68%) levels. Therefore, the fatty acid synthetase alpha subunits of P. patulum and S. cerevisiae obviously contain the same catalytic domains in an identical sequential order.