Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

Specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) have been developed that efficiently inhibit the oncogenic RAF-MEK-ERK pathway. We used a systems-based approach to identify breast cancer subtypes particularly susceptible to MEK inhibitors and to understand molecular mechanisms conferring resistance to such compounds. Basal-type breast cancer cells were found to be particularly susceptible to growth inhibition by small-molecule MEK inhibitors. Activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to MEK inhibition through a negative MEK-epidermal growth factor receptor-PI3K feedback loop was found to limit efficacy. Interruption of this feedback mechanism by targeting MEK and PI3K produced synergistic effects, including induction of apoptosis and, in some cell lines, cell cycle arrest and protection from apoptosis induced by proapoptotic agents. These findings enhance our understanding of the interconnectivity of oncogenic signal transduction circuits and have implications for the design of future clinical trials of MEK inhibitors in breast cancer by guiding patient selection and suggesting rational combination therapies. [Cancer Res 2009;69(2):565-72]

The novel phosphatidylinositol-3-kinase (PI3K) inhibitor PX-866 was tested against 13 experimental human tumor xenografts derived from cell lines of various tissue origins. Mutant PI3K (PIK3CA) and loss of PTEN activity were sufficient, but not necessary, as predictors of sensitivity to the antitumor activity of the PI3K inhibitor PX-866 in the presence of wild-type Ras, whereas mutant oncogenic Ras was a dominant determinant of resistance, even in tumors with coexisting mutations in PIK3CA. The level of activation of PI3K signaling measured by tumor phosphorylated Ser 473 -Akt was insufficient to predict in vivo antitumor response to PX-866. Reverse-phase protein array revealed that the Rasdependent downstream targets c-Myc and cyclin B were elevated in cell lines resistant to PX-866 in vivo. Studies using an H-Ras construct to constitutively and preferentially activate the three best-defined downstream targets of Ras, i.e., Raf, RalGDS, and PI3K, showed that mutant Ras mediates resistance through its ability to use multiple pathways for tumorigenesis. The identification of Ras and downstream signaling pathways driving resistance to PI3K inhibition might serve as an important guide for patient selection as inhibitors enter clinical trials and for the development of rational combinations with other molecularly targeted agents.

Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.

The authors thank Karen Ramirez for her flow cytometric work in the annexin V-propidium iodide cell staining experiments.Address for reprints: Razelle Kurzrock, M.D., Unit 422,