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A hallmark of cancer is reactivation/alteration of pathways that control cellular differentiation during developmental processes. Evidence indicates that WNT, Notch, BMP and Hedgehog pathways have a role in normal epithelial cell differentiation, and that alterations in these pathways accompany establishment of the tumorigenic state. Interestingly, there is recent evidence that these pathways are intertwined at the molecular level, and these nodes of intersection may provide opportunities for effective targeted therapies. This review will highlight the role of the WNT, Notch, BMP and Hedgehog pathways in colon cancer.

The PTEN gene regulates multiple signaling pathways that influence cell proliferation, survival and differentiation. Loss of PTEN expression is closely linked with oncogenesis. Little is known regarding regulation of PTEN gene expression. The PTEN promoter region has been reported and is regulated in part by p53. In a previous study, we found that Notch-1 signaling resulted in increased PTEN protein expression. Herein, we tested the hypothesis that the PTEN gene is a direct target of Notch-1 signal transduction, through binding of the Notch-activated transcription factor CBF-1 to the PTEN minimal promoter. 293 cells expressing constitutively active Notch-1 exhibited increased PTEN gene expression and promoter transactivation. Overexpression of CBF-1 in 293 cells resulted in decreased PTEN gene expression. Mobility shift assays and supershift assays demonstrated that CBF-1 binds to the PTEN minimal promoter. These data indicate that the Notch-1 receptor pathway is a key regulator of PTEN gene transcription.

Mammalian Notch-1 is part of an evolutionarily conserved family of transmembrane receptors best known for involvement in cell fate decisions. Mutations that result in Notch-1 activation result in T-lineage oncogenesis. In other cell lineages, however, studies have indicated that cooperation with cellular signaling pathways, such as Ras, is necessary for Notch-mediated oncogenesis and in some settings, Notch-1 has been reported to function as a tumor suppressor. In order to test the hypothesis that the Notch-1 pathway exhibits cross-talk with Ras/Raf/MEK/ERK, the constitutively active cytoplasmic portion of Notch-1 was introduced into 293 HEK fibroblasts via retroviral transduction. ERK-1,-2 activation was markedly increased in cells expressing constitutively active Notch-1. These cells exhibited a more rounded morphology as compared to 293 cells transduced with an empty vector or parental 293 cells. These observations correlated with decreased total and phosphorylated focal adhesion kinase protein (FAK). Subsequent examination of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) revealed that total and phosphorylated PTEN protein was elevated in cells expressing constitutively active Notch-1. Loss of Akt phosphorylation was also observed in cells bearing activated Notch-1. Two potential binding sites for the Notch effector CBF-1 were identified in the human PTEN promoter sequence. A PTEN promoter luciferase reporter exhibited increased activity in the presence of Notch-1 signaling. These data indicate that Notch-1 can participate in cross-talk with other signaling pathways such as Ras/Raf/MEK/ERK through the regulation of the PTEN tumor suppressor.

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.

v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGFR). EGFR overexpression has been observed in many pathological situations. There is a truncated form of EGFR, referred to as EGFvIII, which resembles v-ErbB in biological properties and is often expressed in certain human tumors. Aberrant EGFR expression in human cancers is often constitutive and may occur in the presence of mutated oncogenes or tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contains the estrogen receptor hormone binding domain (ER) which renders the v-ErbB:ER protein dependent upon beta-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of hematopoietic cells more efficiently than activated v-Ha-Ras, v-Src, Raf or Akt. Abrogation of cytokine-dependence by v-ErbB:ER was not due to the synthesis of autocrine growth factors. Treatment of v-ErbB:ER cells with the EGFR inhibitor AG1478 efficiently induced apoptosis. Induction of apoptosis and prevention of cell cycle progression by the EGFR inhibitor were only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, the other inhibitors suppressed cell cycle progression when the cells were grown in response to v-ErbB:ER or the cytokine interleukin-3. When MEK and either EGFR or PI3K/mTOR inhibitors were added, an enhanced apoptotic response was observed. Thus this conditional ErbB construct is useful to elucidate EGFR signaling and anti-apoptotic pathways in the absence of autocrine cytokine expression.

B-lineage acute leukemia (B-ALL) cells often require stromal cell support for optimal proliferation and apoptotic resistance. In addition, stromal cell contact can promote resistance to chemotherapeutic agents. However, the precise biochemical pathways within the leukemic cell that are activated by the bone marrow microenvironment which result promotion of cell proliferation and apoptotic protection are not fully characterized. We have recently reported that simultaneous inhibition of the MEK and PI3K pathways or the MEK and mTOR pathways promote rapid apoptosis of the stromal cell dependent B-lineage ALL cell line BLIN-2 in the presence of stromal cell support. These data indicated that stromal cell induced apoptotic protection is mediated by PI3K/mTOR and MEK in a mechanism(s) that suggests cross-talk or points of convergence. The EGF receptor (EGFR) has been reported to activate both MEK and PI3K. We report herein that use of the EGFR inhibitor, AG1478, inhibits BLIN-2 survival in the presence of stromal cells. FACS analysis revealed that EGFR is expressed on the surface of BLIN-2 cells. The addition of EGF to BLIN-2 cultures in the absence of stromal cells prolongs BLIN-2 survival. Similarly, introduction of a constitutively active form of EGFR, v-ErbB, into BLIN-2 prolongs the survival of BLIN-2 cells in the absence of stromal cell support. These data provide evidence that stimulation of the EGFR pathway is one mechanism by which the bone marrow microenvironment may contribute to the growth and survival of B-cell acute leukemia.

The recombination activating genes RAG-1 and RAG-2 are highly conserved throughout evolution and are necessary and essential for the DNA rearrangement of antigen-receptor gene segments. These convergently transcribed genes are expressed primarily by developing B and T lineage cells. In addition, recent data suggest that the RAG locus can be reactivated in mouse germinal center B cells. Despite these well-defined patterns of expression, little is known about mechanism(s) regulating transcription of the RAG locus. Experiments with a mouse fibroblast line stably transfected with a genomic fragment of the RAG locus suggest that the intergenic region between RAG-1 and RAG-2 may contain information modulating RAG transcription. In order to begin testing this hypothesis, we have sequenced the 7.0-kb RAG intergenic region of the mouse. The sequence did not contain open reading frames larger than 60 amino acids. Analysis with GCG software identified several potential transcription-factor binding sequences within this region. Many of these are associated with transcriptional regulation of the Ig locus.