Summary The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the inter-conversion of pyruvate and lactate, is upregulated in human cancers and is associated with aggressive tumor outcomes. Here we use a novel inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation in vitro, in vivo, and ex vivo. This was accompanied by re-activation of mitochondrial function in vitro but not in vivo or ex vivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC including cancer stem cell-dependent drug resistant tumors.
IQGAP1 modulates several cellular functions, including cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds ERK and MAPK kinase (MEK) and regulates EGF-stimulated MEK and ERK activity. Here we characterize the interaction between IQGAP1 and B-Raf, the molecule immediately upstream of MEK in the Ras/MAPK signaling cascade. B-Raf binds directly to IQGAP1 in vitro and coimmunoprecipitates with IQGAP1 from cell lysates. Importantly, IQGAP1 modulates B-Raf function. EGF is unable to stimulate B-Raf activity in IQGAP1-null cells and in cells transfected with an IQGAP1 mutant construct that is unable to bind B-Raf. Interestingly, binding to IQGAP1 significantly enhances B-Raf activity in vitro. Our data identify a previously unrecognized interaction between IQGAP1 and B-Raf and suggest that IQGAP1 is a scaffold necessary for activation of B-Raf by EGF.EGF ͉ MAP kinase ͉ signalling T he Ras/Raf/MAPK kinase (MEK)/ERK module is a ubiquitously expressed signaling pathway that conveys mitogenic and differentiation signals from the cell membrane to the interior of the cell (1, 2). This cascade, which is the best studied of the five MAPK pathways, regulates cell growth, proliferation, and differentiation. In response to a stimulus, such as growth factors, cytokines, or hormones, the guanine nucleotide exchange factor Sos induces the exchange of GDP for GTP on Ras, thereby activating Ras. In turn, Ras recruits Raf from the cytosol to the membrane for activation (3, 4). Raf catalyzes the phosphorylation of MEK, which phosphorylates and activates ERK. Activated ERK modulates the function of multiple substrates in all cellular compartments including the nucleus, cytoplasm, and cytoskeleton (2).C-Raf was originally identified as the protein product of the retroviral oncogene v-Raf (5). The Raf family of protein kinases comprises three isoforms, A-Raf, B-Raf, and C-Raf (also known as Raf-1) (4, 6). The Raf proteins share a common architecture, and all function as serine/threonine kinases. Evidence derived by several approaches, including genetic studies in mice, indicates that the proteins have distinct functions. The specific mechanism by which Raf proteins are activated is not known, but oligomerization, binding to other proteins, and multiple phosphorylation events are important (4, 6). Although A-Raf, B-Raf, and C-Raf are all regulated by phosphorylation, the presence of different phosphorylation sites indicates that the proteins can be independently regulated (4). C-Raf is the best characterized and most intensively studied of the Raf isoforms (4). More recently, the identification that B-Raf is an important oncogene (7) has resulted in considerable attention being directed toward B-Raf. Notwithstanding these investigations, much remains to be learned about B-Raf regulation.IQGAP1 is a multidomain molecule that contains several protein-interacting motifs (for reviews, see refs. 8-11). IQGAP1 binds to diverse targets, thereby participating in n...
Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD+ or NADP+. We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP+/NADPH ratio, a drop in ATP, and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease, and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo. Collectively, our data suggest that ME2 is a potential target for cancer therapy.
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