Oncogene (2006) 25, 3247–3257 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis N Moniaux1,4, C Nemos1,4, BM Schmied2, SC Chauhan1, S Deb1, K Morikane2, A Choudhury1, M VanLith2, M Sutherlin2, JM Sikela3, MA Hollingsworth1,2 and SK Batra1,2 1Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA; 2Eppley Institute, University of Nebraska Medical Center, Omaha, NE, USA and 3Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO, USA The 19q13 amplicon in pancreatic cancer cells contains a somes, chromosomal translocations, and gene amplifi- novel pancreatic differentiation 2 (PD2) gene (accession cations, induce a transformed phenotype leading to number AJ401156), which was identified by differential cancer. These genetic alterations constitute key events screening analysis. PD2 is the human homologue of the contributing to tumor progression and metastasis. They RNA polymerase II-associated factor 1 (hPaf1). In yeast, are often stabilized when they confer a growth or Paf1 is part of the transcription machinery, acting as a survivaladvantage to the cells(Lengauer et al., 1998). docking protein in between the complexes Rad6-Bre1, Gene amplification (HSR, homogeneously staining COMPASS-Dot1p, and the phosphorylated carboxyl region and DM, double minute) is one of the most terminal domain of the RNA polymerase II. As such, important mechanisms leading to the alteration of gene Paf1 is directly involved in transcription elongation via expression in solid tumors. The best example studied is histone H2B ubiquitination and histone H3 methylation. the amplification of HER2 on the chromosomal locus The PD2 sequence is highly conserved from Drosophila to 17q12 in primary breast cancer. HER2 is named for humans with up to 98% identity between rodent and human epidermalgrowth factor receptor-2 and belongs human, suggesting the functional importance of PD2/ to the epidermalgrowth factor receptor (EGFR) family hPaf1 to maintain cellular homeostasis. PD2 is a modular of receptor tyrosine kinases (RTKs). In total, 25–30% of protein composed of RNA recognition motif, DEAD- women with breast cancer demonstrate amplification boxes, an aspartic/serine (DS)-domain, a regulator of the and overexpression of the HER2 gene (Slamon et al., chromosome condensation domain and myc-type helix– 1987). Amplification/overexpression of HER2 induces loop–helix domains. Our results further showed that PD2 the phosphorylation of p27Kip (inhibitor of the cdk2) on is a nuclear 80 kDa protein, which interacts with RNA threonine 157 by AKT and its retention in the cytoplasm polymerase II. In addition, we have demonstrated that the (Blain and Massague, 2002; Shin et al., 2002), leading overexpression of PD2 in the NIH 3T3 cells result in enhanced the cells to enter the cell cycle and proliferate. growth rates in vitro and tumor formation in vivo.Altogether, Amplicons often contain several syntenic genes, such this paper presents strong evidence that the overexpression of as an amplification of the 11q13 locus that encompasses PD2/hPaf1 is involved in cancer development. FGF3, FGF4, and CYCD1 (Gaudray et al., 1992) or the Oncogene (2006) 25, 3247–3257. doi:10.1038/sj.onc.1209353; amplification of the locus 12q13 that encompasses published online 20 February 2006 MDM2, CDK4, and GLI (Khatib et al., 1993). We now recognize that amplicons are stabilized in the Keywords: hPaf1; hPAF1 complex; amplicon; pancrea- human genome when they encompass severalgenes that tic cancer provide a selective growth advantage to the cell. In some cases, the stabilization of an amplicon is driven by amplification of nonsyntenic regions such as the co- amplification of the loci 12q13 and 2p24 that encom- passes MYCN (Khatib et al., 1993). Introduction Although DNA amplification is found in a wide range of tumor types, recurrent amplification of a particular The accumulation of genetic alterations in somatic cells, locus seems limited to specific tumors, such as glioma, such as mutations, changes in the number of chromo- breast and ovarian cancers. In an effort to identify differentially expressed genes that may play important Correspondence: Dr SK Batra, Department of Biochemistry and roles in pancreatic tumor growth and progression, Molecular Biology, University of Nebraska Medical Center, 985870 our laboratory pointed out the existence of a double Nebraska MedicalCenter, Omaha, NE 68198-5870, USA. minute amplification corresponding to the chromosomal E-mail: [email protected] 4These authors contributed equally to this work. locus 19q13 (Batra et al., 1991a, b). This genomic Received 25 August 2005; revised 8 November 2005; accepted 10 amplification is now recognized as an underlying cause November 2005; published online 20 February 2006 of many low-frequency genetic events (being amplified Human RNA polymerase II-associated factor 1 N Moniaux et al 3248 in 10–20% of the cases) in pancreatic adenocarcinoma total poly(A+) (Griffin et al., 1994; Ruggeri et al., 1998; Altomare et al., RNA RNA 2003). The 19q13 locus is also amplified in several other cancers such as follicular lymphoma (Werner et al., 1997), Mantle cell lymphoma (Werner et al., 1997), Burkitt’s lymphoma, small-cell lung cancer (Ried et al., CD11/HPAFPanc1 CD11/HPAFPanc1 1994; Petersen et al., 1997), non-small-cell lung cancer (Petersen et al., 1997; Bjorkqvist et al., 1998), breast 2.4 kb carcinoma (Kallioniemi et al., 1994), and uterine cervix cancer (Heselmeyer et al., 1997). Two amplified genes have been characterized at the PD2 19q13 locus, including the ribosomal protein, rpS16, and AKT2 (Batra et al., 1991b; Cheng et al., 1996). AKT2 1.4 kb belongs to a family of three members, AKT1, 2, and 3, which share a strong homology with the PKC and PKA families (Bauer and Baier, 2002). Activation of AKT2 promotes a variety of biological activities involved in tumorigenesis, such as cell survival and cell-cycle progression. In addition, AKT2 overexpression is β-actin reported to be associated with an increase in the aggressiveness of pancreatic cancer cells (Cheng et al., Figure 1 Northern blot analysis of PD2 cDNA with RNA from 1996). For these reasons, AKT2 is considered as the poorly and well-differentiated pancreatic cancer cell lines. Total main target gene associated with the 19q13 amplifi- RNA and purified poly(A) RNA were fractionated by electro- cation. phoresis on 1.2% agarose gelcontaining 0.66 M formaldehyde and transferred onto nitrocellulose via capillary blotting. Lanes 1 and 2 We hypothesized that the stabilization of the 19q13 contained totalRNA from CD11/HPAF and Panc1, respectively. amplicon, detected in 10–20% of the pancreatic Lanes 3 and 4 contained poly(A þ ) RNA from CD11/HPAF and adenocarcinomas, required the presence of another gene Panc1, respectively. The membrane was hybridized with 106 cpm/ favoring tumor development and progression and acting mlof a-32P-labeled PD2/Paf1 cDNA probe. After phospho-imager scanning, the membrane was stripped with 0.1%SDS/0.1 Â SSC synergistically with AKT2. In the present paper, we and re-hybridized with 106 cpm/mlof a-32P-dCTP labeled b-actin describe the identification and characterization of such a probe. novelgene, pancreatic differentiation factor 2 ( PD2). We show that the PD2 gene product is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1), a key component of a multifunctional The chromosomal localization of the PD2 gene was transcriptional complex that is involved in the regula- determined by screening both the CEPH megabase-insert tion of gene transcription. The domain-structure of PD2 YAC DNA and the Coriell human X rodent somatic cell is conserved in evolution from yeast to human with up hybrid DNA pools. PD2 was mapped to the short arm to 98% identity between rodent and human. We of chromosome 19 between the q13–qter regions. The demonstrate clearly that PD2/hPaf1 possesses trans- NCBI (NationalCenter of BiotechnologyInformation) forming activity both in vitro and in vivo. human genome resources database was used to resolve its precise location. The PD2 gene was located on chromosome 19 in the q13.2 (loc126275) region, oriented from centromere to telomere, between the Results IgG binding protein gene (FcGBP) and the zinc-finger protein 36 gene (ZFP36). Interestingly, both PD2 and Identification of the human homologue of yeast Paf1 AKT2 presented the same chromosomal localization, as part of the 19q13 amplicon 19q13.2 (Figure 2). Since AKT2 is known to be present To identify markers of differentiation for pancreatic in an amplicon, we hypothesized that PD2 might be cancer, single-stranded cDNA probes synthesized from carried on the same chromosomalamplification. South- both poorly and well-differentiated tumor cells, Panc1 ern blot analysis was performed on Panc1 and CD11/ and CD11/HPAF, respectively, were used to screen a HPAF cells (Figure 3), and revealed that Panc1 cells lgt11 phage cDNA library of Panc1 cells (Batra et al., contained a 30-fold gene amplification of PD2 as 1991a, b). In total, 17 clones were isolated, subcloned compared to CD11/HPAF. The ubiquitous expression into the pBluescript vector, and sequenced. Among the of PD2/hPaf1 in human, as revealed by blast scan using clones isolated, three were previously reported as the partialPD2 cDNA sequence, and presence of DNA- human ribosomalprotein S16 (Batra et al., 1991b), the and RNA-binding domains within its sequence, led us to human ribosomalprotein rpL17 (Batra et al., 1991a), hypothesize that PD2 might act in concert with AKT2 and AKT2 (Cheng et al., 1996). Another clone from this for the stabilization of the 19q13 amplification. screening, called PD2, showed an overexpression of To validate this hypothesis, we decided to extend and approximately 30-fold in Panc1 as compared to CD11/ fully characterize the PD2 partial sequence.