Upper picture is a confocal image in the z plane of a green fluorescent cell protruding invadopodia membranes through the microporus filter (not visible). Lower picture is a cell protruding its invadopodia through a 3.0 um pore to the lower surface. Invadopodia are stained with rodamine-phalloidin to visualize the f-actin cytoskeleton.
 

Dr. Klemke's research program employs unique proteomic, phosphop­roteomic, genomic and bioinformatics methodologies to identify novel phospho­protein/­kinase biomarkers (fingerprints) and map global kinase signaling networks that control cell polarization, immune cell homing, cell migration, proliferation, drug resistance, and cancer cell metastasis. To obtain the 'fingerprint' of metastatic cells, our lab has pioneered the development of important new subcellular membrane purification methods using microporous filters to uncover thousands of unique protein and gene signatures, which drive the metastatic behavior of cancer and immune cells. These molecular 'fingerprints' and their annotated signaling networks are being used as biomarkers to detect metastatic cells in the body and to evaluate the efficacy of novel anti-tumor and anti-metastatic agents using preclinical animal models of cancer.

Project 1: Invadopodia proteomics and metastatic signature identification: Pseudopodia and invadopodia are membrane projections made by invasive migrating cells that facilitate extracellular matrix degradation and migration through complex tissues in the body (Figure1). Cell migration during cancer metastasis requires morphological polarization characterized by formation of a leading pseudopodium (PD) at the front and a trailing rear at the back. This process is controlled by the spatial and temporal organization of complex protein signaling networks that partition to the front or back of the cell. Reversible protein phosphorylation on serine, threonine, or tyrosine amino acid residues is a major mechanism utilized by migratory cells to coordinate the localization, activation, stability, and assembly of macromolecular signaling complexes. However, aberrant regulation of protein localization and protein phosphorylation in cancer cells contributes to the processes of cell invasion, migration and metastasis. Using unique invadopodia/pseudopodia purification methods developed in the Klemke Lab, we have applied global proteome profiling in combination with quantitative phosphoproteomics and bioinformatics approaches for comparative analysis of the cell rear (cell body, CB) and invadopodia proteomes of invasive migrating cells. The spatial relationship of greater than 4000 proteins and hundreds of distinct sites of phosphorylation can be mapped revealing networks of signaling proteins that partition to the invadopodium and/or the cell body compartments. This powerful approach as revealed unique proteins and networks of phosphoprotein signatures that endow these cells with metastatic abilities. These protein signatures have important diagnostic and prognostic value, and are possible new therapeutic targets. Important new metastatic proteins have been identified and are being functionally characterized at the gene and protein level in the lab, including Lasp-1, PEAK1 tyrosine kinase, and eIF5A. Additional novel metastatic signatures and new phosphorylation sites have been identified and are ready for functional testing in cell-based and preclinical animal models of cancer. Computational and informatics analyses are also underway to understand how these metastatic signatures and kinase substrate interactions are integrated at the network level to control cell invasion and metastasis.

Project 2: PEAK1 and Cancer Progression. Using the invadopodia purification technology developed in our laboratory, we discovered pseudopodia-enriched atypical kinase one (PEAK1), a catalytically active tyrosine kinase that associates with the actin cytoskeleton and regulates cell migration and proliferation. PEAK1 is also an important scaffolding protein that couples integrin and growth factor receptor signaling to the cytoskeleton to control cell movement and proliferation. Figure one

Project 2a. Role of PEAK1 tyrosine kinase in pancreatic cancer formation and metastasis. Work pioneered in our lab has shown that PEAK1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) where it regulates tumor cell proliferation, metastasis and chemoresistance (Figure 1). Emerging work in the field indicates that this newly discovered kinase is a critical player in several human cancers including PDAC, colon, and breast cancer. To directly address the function of PEAK1 in normal physiology and cancer a conditional PEAK1 knockout animal has been generated in our lab using Cre-lox technology. Future projects will involve breeding the PEAK1 knockout mice to genetically engineered mouse models of PDAC to determine how PEAK1 contributes to cancer formation, progression and drug resistance. The normal physiological function of PEAK1 will also be investigated in these mice.

Project 2b. Dissecting the signal transduction role of PEAK1

The goal of these studies is to determine precisely how PEAK1 signaling (Figure 1) couples to the cell migration and proliferation machineries of normal and malignant cells. For these studies, we are using genetic engineering and site directed mutagenesis to perturb key signaling domains and phosphorylation sites in PEAK1. The mutant forms of PEAK1 are then tested for their ability to transmit various migration and proliferation signals within the cell's interior. Critical domains in PEAK1 that modulate src kinase signaling, adaptor protein binding, and kinase activity have been identified and functionally validated. PEAK1 signaling functions are also being investigated at the network level using PEAK1 knockout/knockin cells and powerful proteomic and phosphoproteomic profiling methods combined with computer-based informatics programs. Additional biochemical, mutagenesis, and proteomic works are in progress to identify specific PEAK1 binding partners and key effector pathways.

Project 2C. Role of PEAK1 in developmental and tumor-induced angiogenesis. New blood vessel formation (angiogenesis) is required for the sustained growth of primary and secondary metastatic tumor growth. These vessels provide life sustaining oxygen and nutrients that fuel new tumor growth. Therefore, inhibiting the process of tumor-induced angiogenesis is being intensely investigated as a possible means to inhibit tumor growth. Work in our lab has show n that PEAK1 tyrosine kinase plays a critical role in new vessel growth. In fact, Knockout of PEAK1 in zebrafish using TALEN technology (Transcription Activator-Like Effector Nucleases) or knockdown by specific PEAK1 morpholinos (MO) induced severe pericardial edema, blood-pooling defects, and disrupted the formation of intersegmental (ISV) and subintestinal vessels (SIV) (Figures 1 and 2). High-resolution intravital time-lapse imaging of the developing vasculature revealed that PEAK1 depletion inhibits both endothelial cell proliferation and migration of ISVs. Co-injection of suboptimal doses of PEAK1 and VEGF MO, which does not inhibit vessel development alone, induced a robust inhibition of ISV angiogenesis, suggesting PEAK1 has a synergistic role with VEGF. In further support of these findings, depletion of PEAK1 in HUVECs inhibits tube formation and vessel sprouting on fibrin-coated beads induced by VEGF-A. In addition, treatment of HUVECs with VEGF-A promoted phosphorylation of tyrosine 665 of PEAK1, a key Src kinase SH2 adaptor binding site. Finally, we found that PEAK1 expression is highly increased in the endothelium of neovessels of human tumor tissue samples compared to the quiescent endothelium of normal vessels suggesting that PEAK1 plays a role in tumor-induced angiogenesis. Together our findings indicate that PEAK1 is a novel tyrosine kinase necessary for angiogenesis. These findings also suggest that PEAK1 is a good therapeutic target to inhibit cancer, since it plays a dual role in tumor formation and tumor-induced angiogenesis. Signal transduction work is now underway to understand how VEGF receptor signaling activates PEAK1 to control endothelial cell proliferation and migration. We also have plans to investigate the role of PEAK1 in other vascular related diseases including retinopathy and wound healing.

Project 2D. Role of PEAK1 in cancer stem cell propagation and differentiation. Neoplastic tissues are believed to contain a subpopulation of stem-like cells with self-renewing capabilities (referred to cancer stem cells, CSCs). These cells are believed to be the metastatic culprits and the underlying cause of patient deaths due to relapse after chemotherapy and radiation. Therefore, new therapeutic approaches and strategies are sorely needed to target these highly resistant cells. Work in our lab has shown that PEAK1 is a new tyrosine kinase that controls the stem cell transcriptional machinery present in CSCs. Using a genetic PEAK1 knockout/Knockin approach, we have shown that PEAK1 signaling is the major driving component of a feed-forward transcriptional loop that controls the expression of the known stem cell regulators Oct-4 (Figure 1), c-myc, Soxs2, KLF4, Yap1, and Nanog. Perturbation of PEAK1 function inhibits the feed-forward loop, preventing tumor initiation and formation in vitro and in vivo. We are currently investigating in detail how extracellular cues in the tumor microenvironment integrate PEAK1 signaling with the CSC transcriptional program to drive tumor initiation and metastasis.