Oncogene (2004) 23, 1354–1363 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc Overexpression of the amplified Pip4k2b gene from 17q11–12 in breast cancer cells confers proliferation advantage Shiuh-Wen Luoh*,1,2,4, Natarajan Venkatesan3 and Reshimi Tripathi1 1Department of Medicine, Division of Hematology and Oncology, University of Cincinnati, College of Medicine, Cincinnati, OH 45267, USA; 2Cincinnati Veterans’ Affairs Medical Center Cincinnati, Cincinnati, OH 45220, USA; 3Division of Hematology and Oncology, Department of Medicine, UCLA Medical Center, Los Angeles, CA 90095, USA Gene amplification is common in solid tumors and is amplification maximum distinct from that of HER-2/Neu associated with adverse prognosis, disease progression, and serve as an independent target for amplification and and development of drug resistance. Asmall segment from selective retention. Pip4k2b amplification is associated chromosome 17q11–12 containing the HER-2/Neu gene is with overexpression at the RNAand protein level in breast amplified in about 25% of breast cancer. HER-2/Neu cancer cell lines. Stable expression of Pip4k2b in breast amplification is associated with adverse prognosis and cancer cell lines with and without HER-2/Neu amplifica- may predict response to chemotherapy and hormonal tion increases cell proliferation and anchorage-indepen- manipulation. Moreover, HER-2/Neu amplification may dent growth. The above observations implicate Pip4k2b in select patients for anti-HER-2/Neu-based therapy with the development and/or progression of breast cancer. Our Herceptin. We and others recently described a common study suggests that Pip4k2b may be a distinct target for sequence element from the HER-2/Neu region that was gene amplification and selective retention from 17q11–12. amplified in breast cancer cells. In addition, most, if not Oncogene (2004) 23, 1354–1363. doi:10.1038/sj.onc.1207251 all, of the amplified genes from this region display Published online 22 December 2003 overexpression. This raises the intriguing possibility that genes immediately adjacent to HER-2/Neu may influence Keywords: tumor biology; breast cancer; gene the biological behavior of breast cancer carrying HER-2/ amplification; gene expression; HER-2/Neu Neu amplification and serve as rational targets for therapy. By extracting sequence information from public databases, we have constructed a contig in bacterial artificial chromosomes (BACs) that extends from HER-2/ Introduction Neu to a phosphotidylinositol phosphate kinase (PIPK), Pip4k2b from 17q11–12. Although a role of PI-3-kinase Gene amplification is common in solid tumors and is and AKT in cancer biology has been previously described, associated with adverse prognosis, disease progression, PIPK has not been previously implicated. We show that and development of resistance to chemotherapy (Save- Pip4k2b, initially known as Pip5k2b, is amplified in a lyeva and Schwab, 2001). Amplification of N-Myc in subset of breast cancer cell lines and primary breast neuroblastoma (Seeger et al., 1985; Rubie et al., 1997) cancer samples that carry HER-2/Neu amplification. Out and amplification of HER-2/Neu in breast cancer are of eight breast cancer cell lines with HER-2/Neu associated with shortened survival (Slamon et al., 1987, amplification, three have concomitant amplification of 1989). Amplification of the DHFR gene enables cancer the Pip4k2b gene – UACC-812, BT-474 and ZR-75-30. cells to acquire resistance to the chemotherapeutic Similarly, two out of four primary breast tumors with agent, methotrexate (Savelyeva and Schwab, 2001). HER-2/Neu amplification carry Pip4k2b gene amplifica- Breast cancer with HER-2/Neu amplification may tion. Intriguingly, one tumor displays an increase in the exhibit a relative resistance to nonanthracycline-based gene copy number of Pip4k2b that is significantly more therapy but display retained or increased sensitivity to than that of HER-2/Neu. Moreover, dual color FISH anthracycline-based treatment (Muss et al., 1994; Paik reveals that amplified Pip4k2b gene may exist in a distinct et al., 1998; Thor et al., 1998). Amplification of structure from that of HER-2/Neu in ZR-75-30 cell line. chromosome 17q11–12 involving the HER-2/Neu gene These studies suggest that Pip4k2b may reside on an has been described in breast (Slamon et al., 1987), ovarian (Slamon et al., 1989), head and neck (Press et al., *Correspondence: S-W Luoh, Division of Hematology and Oncology, 1994), prostate (Ross et al., 1997), endometrial (Rolitsky Oregon Health Sciences University, Portland VA Medical Center, 3710 et al., 1999), gastric and esophageal cancers (Ishikawa SW US Veterans Hospital Road, RAD 47, Portland, OR 97239, USA; et al., 1997; Nakajima et al., 1999; Nessling et al., 1998; Fax: 503-273-5351 Brien et al., 2000; Walch et al., 2000). As is the case with 4Supported in part by American Cancer Society, Ohio Chapter, Ohio Cancer Research Associates, and VA Merit Award gene amplification in general, an increase in the copy Received 26 June 2003; revised 4 September 2003; accepted 29 September number of HER-2/Neu gene leads to overexpression of 2003 HER-2/Neu transcript and protein. Pip4k2b amplification in breast cancer S-W Luoh et al 1355 Retention of amplified sequences in cancer cells In this study, we show that Pip4k2b is amplified and indicates evolutionary advantage conferred by these overexpressed in a subset of breast cancer cell lines. amplified genes. Gene amplification may involve vari- Amplification of Pip4k2b is associated with overexpres- able amount of sequence. At one extreme, the amplified sion on the transcript and protein level. An increase in sequence involving N-Myc in some neuroblastoma cells the gene copy number of Pip4k2b that is significantly is pruned to a very limited segment of which N-Myc may more than that of HER-2/Neu has been observed in one be the only contributing genetic element (Amler and primary breast tumor. Moreover, dual color fluores- Schwab, 1989). On the other hand, amplification of cence in situ hybridization (FISH)reveals that amplified chromosome 11q13 may involve large, discontinuous Pip4k2b gene may exist in structure not accompanied by blocks of chromosomal DNA (Gaudray et al., 1992; that of HER-2/Neu in ZR-75-30 breast cancer cell line. Szepetowski et al., 1993). The extent of the sequence These studies suggest that Pip4k2b may fall on an that is amplified appears to be distinct among various amplification maximum distinct from that of HER-2/ tumor types. The pattern of overexpression of genes from Neu and serve as an independent target for amplification the amplified segment differs as well (Theillet et al., 1990; and selective retention. Overexpression of Pip4k2b in Lammie et al., 1991; Lammie and Peters, 1991; Proctor breast cancer cell lines confers proliferation advantage et al., 1991; Szepetowski et al., 1992; Roelofs et al., 1993; and promotes anchorage-independent growth. Pip4k2b Bringuier et al., 1996). This nonuniformity provides may therefore play important roles in the development evidence that distinct gene(s)are targets of 11q13 and/or progression of breast cancer. amplification in various tumor types. Similar complexity has been described for amplification events involving 17q22–23. Several specific and independent amplification Results maxima from this region were identified in breast cancer cell lines and breast tumors (Barlund et al., 2000; Wu A 17q11–12 contig from HER-2/Neu to Pip4k2b et al., 2000; Monni et al., 2001; Wu et al., 2001). The gene content of the HER-2/Neu amplified By extracting sequence information from the public sequences has recently been elucidated. Multiple genes databases, we assembled a contig in BACs that extended from the chromosomal segment containing HER-2/Neu from Hs.374824 through the HER-2/Neu gene to a lipid gene are coamplified with HER-2/Neu (Kauraniemi kinase, Pip4k2b from chromosome 17q11–12 (Figure 1). et al., 2001; Luoh, 2002). Interestingly, there is near Overlap was established based on identity of sequenced perfect correlation between increased gene dosage and portions of different BACs. This was verified with overexpression for these genes in breast cancer cell lines multiple PCR assays using primers derived from the (Kauraniemi et al., 2001; Luoh, 2002). This observation regions of overlap (data not shown). Dual color FISH raises the intriguing possibility that overexpression of using two differentially labeled BAC probes derived multiple coamplified genes, rather than amplification of from this region revealed colocalization of hybridization HER-2/Neu alone, may contribute to the development signals in breast cancer cells (not shown). A similar and/or progression of breast cancer that carries 17q11– contig using many of the BACs reported here has 12 amplification. Moreover, these genes may serve as recently been reported by NCBI (NT_010755), in additional targets for therapy for this subset of breast confirmation of our result. The whole region has been cancer patients who have a more aggressive form of completely sequenced. The distance between Hs.374824 disease. and HER-2/Neu is about 220 kb and that between HER- By extracting sequence information from public data- 2/Neu and Pip4k2b is about 900 kb. bases, we have constructed a contig in bacterial artificial chromosomes (BACs)that extends from HER-2/Neu to a phosphotidylinositol phosphate kinase (PIPK), Pip4k2b from 17q11–12. Initially known as Pip5k2b it is now reclassified as Pip4k2b (Rameh et al., 1997). The importance of phosphoinositides (PI)as lipid signaling molecules in eukaryotic cells has been well character- ized. A number of phosphotidylinositol phosphate Figure 1 Physical linkage of HER-2/Neu and Pip4k2b.A kinases have been described that can phosphorylate chromosomal segment from 17q11–12 extending from HER-2/ different positions of the inositol ring leading to the Neu to Pip4k2b was cloned in a set of overlapping BACs. A total of formation of PIP2 and PIP3 that have various signaling 13 BACs were used to assemble this contig.