CREB is a transcription factor that functions in glucose homeostasis, growth factor-dependent cell survival, and memory. In this study, we describe a role of CREB in human cancer. CREB overexpression is associated with increased risk of relapse and decreased event-free survival. CREB levels are elevated in blast cells from patients with acute myeloid leukemia. To understand the role of CREB in leukemogenesis, we studied the biological consequences of CREB overexpression in primary human leukemia cells, leukemia cell lines, and transgenic mice. Our results demonstrate that CREB promotes abnormal proliferation and survival of myeloid cells in vitro and in vivo through upregulation of specific target genes. Thus, we report that CREB is implicated in myeloid cell transformation.

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

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Prior studies have identi®ed Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The ®rst transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in nontransgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTVFgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at ®ve and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.

Leukemia is a result of accumulating genetic alterations. The collaboration of mutations that offer survival and proliferative signals, together with mutations that result in lack of differentiation, is thought to cause a leukemic phenotype. The cyclic-AMP Response Element Binding Protein (CREB) is a transcription factor that is known to be a downstream component of the GM-CSF and IL-3 signaling pathways. We previously showed that CREB is overexpressed in blast cells from patients with acute leukemias. In this paper, we review the role of CREB in hematopoiesis, cell proliferation and acute leukemias.

We have used mouse mammary tumor virus (MMTV) infection of Wnt-1 transgenic mice to accelerate mammary tumorigenesis and to molecularly tag insertionally activated proto-oncogenes that cooperate oncogenically with Wnt-1 (G. M. Shackleford, C. A. MacArthur, H. C. Kwan, and H. E. Varmus, Proc. Natl. Acad. Sci. USA 90:740-744, 1993). Here we report the identification and characterization of a 31-kb genomic locus that contains clonal MMTV integrations in 8 of 80 mammary tumors from MMTV-infected Wnt-1 transgenic mice. Two genes were identified within this locus, one of which was transcriptionally activated by MMTV insertions. This activated gene is identical to androgen-induced growth factor (AIGF/Fgf-8) (A. Tanaka, K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto, Proc. Natl. Acad. Sci. USA 89:8928-8932, 1992), the eighth member of the fibroblast growth factor (FGF) family. Transcriptional activation of Fgf-8 was found in all tumors with MMTV insertions in this locus. Fgf-8 mRNA was absent in normal mammary glands and was detected only in adult testis and ovary and in midgestational embryos. The sequences of Fgf-8 genomic and cDNA clones revealed five coding exons, in contrast to the three coding exons found in other FGF genes. cDNAs encoding three isoforms of the FGF-8 protein were isolated. The three corresponding mRNAs resulted from the alternative use of two 5 splice sites and two 3 splice sites for the second and third exons, respectively. These results implicate Fgf-8 as the third FGF gene found to cooperate with Wnt-1 in MMTV-induced murine mammary tumorigenesis, suggesting that FGFs and Wnts are strong collaborators in this process.