Elevated ITGA2 expression promotes collagen type I-induced clonogenic growth of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) arises along the peripheral bile ducts and is often accompanied by a tumor microenvironment (TME) high in extracellular matrices (ECMs). In this study, we aimed to evaluate whether an ECM-rich TME favors iCCA progression. We identified ITGA2, which encodes collagen-binding integrin α2, to be differentially-expressed in iCCA tumors compared with adjacent normal tissues. Elevated ITGA2 is also positively-correlated with its ligand, collagen type I. Increased ITGA2 expression and its role in collagen type I binding was validated in vitro using four iCCA cell lines, compared with a non-cancerous, cholangiocyte cell line. Robust interaction of iCCA cells with collagen type I was abolished by either ITGA2 depletion or integrin α2ß1-selective inhibitor treatment. In a phenotypic study, collagen type I significantly enhances clonogenic growth of HuCCA-1 and HuCCT-1 cells by three and sixfold, respectively. Inhibition of integrin α2 expression or its activity significantly blocks collagen type I-induced colony growth in both cell lines. Taken together, our data provide mechanistic evidence that collagen type I promotes growth of iCCA colonies through integrin α2 suggesting that the collagen type I-integrin α2 axis could be a promising target for cancer prevention and a therapeutic opportunity for this cancer.

EGFR Signals through a DOCK180-MLK3 Axis to Drive Glioblastoma Cell Invasion.

A hallmark of glioblastoma (GBM) tumors is their highly invasive behavior. Tumor dissemination into surrounding brain tissue is responsible for incomplete surgical resection, and subsequent tumor recurrence. Identification of targets that control GBM cell dissemination is critical for developing effective therapies to treat GBM. A majority of GBM tumors have dysregulated EGFR signaling, due most frequently to EGFR amplification or the presence of a constitutively active EGFRvIII mutant. Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that can activate multiple MAPK pathways. In this study, evidence is provided that MLK3 is essential for GBM cell migration and invasion, and that an MLK inhibitor blocks EGF-induced migration and invasion. MLK3 silencing or MLK inhibition blocks EGF-induced JNK activation, suggesting that MLK3-JNK signaling promotes invasion of GBM cells. Mechanistically, it is demonstrated that DOCK180, a RAC1 guanine nucleotide exchange factor (GEF) overexpressed in invasive GBM cells, activates the MLK3-JNK signaling axis in a RAC1-dependent manner. In summary, this investigation identifies an EGFR-DOCK180-RAC1-MLK3-JNK signaling axis that drives glioblastoma cell migration and dissemination.Implications: On the basis of these findings, MLK3 emerges as a potential therapeutic target for the treatment of glioblastoma. Mol Cancer Res; 15(8); 1085-95. ©2017 AACR.

MLK3 Signaling in Cancer Invasion.

Mixed-lineage kinase 3 (MLK3) was first cloned in 1994; however, only in the past decade has MLK3 become recognized as a player in oncogenic signaling. MLK3 is a mitogen-activated protein kinase kinase kinase (MAP3K) that mediates signals from several cell surface receptors including receptor tyrosine kinases (RTKs), chemokine receptors, and cytokine receptors. Once activated, MLK3 transduces signals to multiple downstream pathways, primarily to c-Jun terminal kinase (JNK) MAPK, as well as to extracellular-signal-regulated kinase (ERK) MAPK, P38 MAPK, and NF-κB, resulting in both transcriptional and post-translational regulation of multiple effector proteins. In several types of cancer, MLK3 signaling is implicated in promoting cell proliferation, as well as driving cell migration, invasion and metastasis.

Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3ß and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.