Signaling Cascades Regulating Cell Growth, Metastasis, or Cell Death
Cancer is characterized by loss of normal cellular growth control. Intracellular signal transduction pathways are critical to the proper interpretation and integration of growth regulatory stimuli, and intricate mechanisms have evolved for ensuring the fidelity of cell replication. Small changes that alter the magnitude of these signals can significantly impact cellular outcomes. Thus, elucidating the nature of these signaling pathways and how they are modulated is central to understanding cell cycle control and the maintenance of genomic integrity. The focus of our laboratory is to determine the critical mechanisms that regulate cell growth and differentiation in response to growth factor or oncogenic stimulation and identify key targets for therapeutic intervention.
One of the main players in the regulation of cell growth is the MAP kinase cascade, an evolutionarily conserved signaling pathway that responds to a number of extracellular stimuli. The extracellular signal regulated kinases (ERKs) are a subfamily of MAP kinases that are activated via a cascade involving Ras, Raf kinase, and MEK/ERK kinase (MEK). Activation of the MAP kinase pathway is tightly controlled, and Raf-1 activation is a key regulatory step in this process. Raf-1 is also regulated by a number of proteins that modulate its activity, leading to different physiological outcomes. One of the regulators of Raf-1 signaling is Raf Kinase Inhibitory Protein (RKIP), also known as Phosphatidylethanolamine Binding Protein 1 (PEBP1). RKIP inhibits MAP kinase signaling in part by binding to Raf-1, preventing Raf-1 phosphorylation at activating sites. RKIP depletion increases the amplitude and dose response of MAP kinase activation and DNA synthesis in EGF-treated cells. RKIP is a ubiquitously expressed and highly conserved protein, and many of its homologs regulate growth and differentiation signaling pathways.
1.RKIP Integrates many different signaling pathways
Work from our laboratory and others suggests that RKIP maintains homeostasis in cells, and its function is highly responsive to its phosphorylation state. We showed that RKIP is phosphorylated on S153 by Protein Kinase C (PKC), causing dissociation of RKIP from Raf-1 (Corbit et al., 2003) and Raf-1 activation (Trakul et al., 2005). Phosphorylated RKIP inhibits G protein coupled Receptor Kinase 2 enhancing G protein-coupled receptor signaling cascades such as those mediated by Protein Kinase A (PKA). These studies indicate that RKIP acts as an endogenous modulator of the MAPK and PKA signaling cascades, limiting the response of the cell to growth factor stimuli. We have also shown that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf/MEK/ERK cascade, and demonstrated that small changes in the MAP kinase pathway can profoundly impact the fidelity of the cell cycle. The precise mechanism by which RKIP integrates phosphorylation by different kinases into a single functional output is under study.
A member of an evolutionarily conserved family of over 400 members, RKIP is regulated differently than other proteins with ligand-binding pockets. Using approaches ranging from NMR to animal knockout models, we have shown that the ligand-binding pocket region of RKIP regulates its inhibition of Raf-1 by altering the ability of other kinases to bind and phosphorylate RKIP. Future structural studies of RKIP are focused on the molecular interactions that govern the regulatory functions of RKIP and other members of the evolutionarily conserved PEBP family.
2. RKIP and cancer.
As a regulator of key kinase signaling cascades including MAP kinase, NFkB, and GPCRs, RKIP can play a critical role in progression to diseases such as cancer. Clearly cancer is a complex problem that involves early events such as tumor initiation, epithelial-mesenchymal transition (EMT), migration, invasion, and intravasation into blood vessels as well as later events such as extravasation from blood vessels, mesenchymal-epithelial transition (MET), and colonization at other sites within the body including bone and lung.Identifying key genes and signaling pathways that regulate these processes would enable not only more accurate prognoses but also identify important mediators that could serve as therapeutic targets.
One approach is to characterize the targets of tumor metastasis suppressors. As inhibitors of metastatic progression and colonization, metastasis suppressors represent important markers for prognosis and potential effectors of therapeutic treatment. Raf Kinase Inhibitory Protein (RKIP) was initially implicated as a suppressor of metastasis in a murine model using androgen-independent prostate tumor cells. We recently demonstrated that expression of RKIP inhibits intravasation and bone metastasis in mice using human breast cancer cells. Our results identified a pathway whereby RKIP negatively regulates Raf-1/MAPK activity, leading to inhibition of Myc and LIN28 as well as induction of let-7, an evolutionarily conserved microRNA whose processing is regulated by LIN28 (9)(see Fig. 1).
Despite the role of let-7 as a suppressor of TICs and breast cancer metastasis, the downstream signaling targets are largely unknown. To identify putative downstream targets of RKIP and let-7 that mediate bone metastasis, we recently used a novel approach based on analysis of gene expression arrays derived from patient tumors. We negatively correlated expression of putative let-7 targets with RKIP expression in >1200 human breast tumors using gene set analysis (GSA). We used a similar approach to test the hypothesis that bone metastasis signature (BMS) genes are regulated by RKIP. Gene signatures for breast cancer metastases to bone and lung have been previously described based upon specific enrichment in cells that preferentially metastasize to these sites in xenograft models. Our results identified a novel RKIP/let-7-regulated signaling cascade that we validated experimentally and clinically (Figure 1). This approach was very successful and enabled us to predict individuals at risk for breast metastasis due to dysregulation of this RKIP signaling cascade. We plan to continue to use this GSA-based strategy to identify genes that regulate tumor-initiating and metastatic cells.
Both oxidative stress and epigenetic reprogramming have recently gained much attention as key regulators of progression to cancer. Two genes that we identified as key targets of RKIP/let-7 that promote breast cancer metastasis are BACH1, a transcription factor that has been implicated in the control of the redox state, and HMGA2, a chromatin remodeling factor. We are currently investigating the mechanisms by which BACH1 and HMGA2 promote tumorigenesis and metastasis.
Tumor interactions with the surrounding stromal cells in the microenvironment are major determinants of the tumor cell’s ability to survive and undergo invasion and metastasis. Transcriptional analyses have revealed that stromal cells undergo alterations in gene expression following exposure to tumor cells..In collaboration with the laboratory of Yoav Gilad, we are developing a novel approach based on next generation RNA sequencing to identify, with high precision, genes involved in the interplay between the tumor cell and its microenvironment. These studies will enable us to elucidate differences in tumor-stromal signaling between invasive and metastatic breast tumors versus noninvasive, RKIP-expressing breast tumors in order to identify and understand the nature of the reprogramming between the tumor and its microenvironment.
3. Other regulators of tumorigenesis.
Maintenance of tumors requires progression through the cell cycle, and access to nutrients through angiogenesis. We have shown that Protein kinase C induces cyclin E through a feed forward loop involving suppression of microRNA15. In order to understand the importance of this feed forward network, we are currently modeling the induction of cyclin E during the cell cycle. We have also shown, in collaboration with the Tao Pan laboratory, that tRNAs are elevated in tumors; ribosome profiling should reveal the specific transcripts that are translated in response to elevated tRNA expression. Furthermore, tRNAs can be engineered as "killer tRNAs" to eliminate tumor cells. Finally, we have identified a Rap-regulated signaling cascade that controls angiogenesis through a novel interplay between tumor and endothelial cells. Each of these signaling cascades represents a different hallmark of cancer.