Syntaphilin controls a mitochondrial rheostat for proliferation-motility decisions in cancer.

Tumors adapt to an unfavorable microenvironment by controlling the balance between cell proliferation and cell motility, but the regulators of this process are largely unknown. Here, we show that an alternatively spliced isoform of syntaphilin (SNPH), a cytoskeletal regulator of mitochondrial movements in neurons, is directed to mitochondria of tumor cells. Mitochondrial SNPH buffers oxidative stress and maintains complex II-dependent bioenergetics, sustaining local tumor growth while restricting mitochondrial redistribution to the cortical cytoskeleton and tumor cell motility. Conversely, introduction of stress stimuli to the microenvironment, including hypoxia, acutely lowered SNPH levels, resulting in bioenergetics defects and increased superoxide production. In turn, this suppressed tumor cell proliferation but increased tumor cell invasion via greater mitochondrial trafficking to the cortical cytoskeleton. Loss of SNPH or expression of an SNPH mutant lacking the mitochondrial localization sequence resulted in increased metastatic dissemination in xenograft or syngeneic tumor models in vivo. Accordingly, tumor cells that acquired the ability to metastasize in vivo constitutively downregulated SNPH and exhibited higher oxidative stress, reduced cell proliferation, and increased cell motility. Therefore, SNPH is a stress-regulated mitochondrial switch of the cell proliferation-motility balance in cancer, and its pathway may represent a therapeutic target

Novel roles of the RB-pathway in human cancers: Modifying disease progression and establishing new therapeutic opportunities

Deregulation of the retinoblastoma (RB) tumor suppressor pathway, a critical negative regulator of cell cycle control and proliferation, is observed in a wide variety of human cancers. Emerging studies have implicated RB protein function not only in cell proliferation but also in other essential cellular processes such as differentiation and chromosomal instability, among others. However, how deregulation of the RB-pathway directly impacts certain critical aspects of tumorigenesis and disease progression still remains an enigma. Herein, using breast cancer models, we have identified novel implications of RB-pathway deregulation that contribute to disease progression. RB loss in cooperation with epidermal growth factor 2 (ErbB2), a prominent oncogene activated in breast cancer, promotes structural and organizational changes in mammary epithelial cells correlating with an epithelial-to-mesenchymal transition gene signature, and ultimately an invasive phenotype in mammary lesions in vivo. Additionally, due to the clear importance of RB in suppressing tumorigenesis in multiple tissue types, we examined the efficacy of using a small molecule CDK4/6 inhibitor (PD-0332991) to activate RB function as a potential therapeutic modality in both early stage breast cancer and advanced liver cancer models. CDK4/6 inhibition was observed to be highly effective at inducing a potent cytostatic response in both model systems. Importantly, we demonstrate that the efficacy of PD-0332991 is dependent on the expression of multiple key players in cell cycle control aside from RB itself, including the other pocket proteins (p107/p130), p16 ink4a and Cyclin D1. Together these studies demonstrate the utility of RB-pathway status as both a predictor for invasive disease and a novel therapeutic target

RB and p53 cooperate to prevent liver tumorigenesis in response to tissue damage.

The tumor suppressors retinoblastoma (RB) and p53 are important regulators of the cell cycle. Although human cancer cells inactivate RB and p53 by many mechanisms, the cooperative roles of these proteins in tumorigenesis are complex and tissue specific. We analyzed the cooperation of RB and p53 in liver development and pathogenesis of hepatocellular carcinoma. Spontaneous and carcinogen-induced (diethylnitrosamine) tumorigenesis were studied in mice with liver-specific deletions of Rb and/or p53 (Rbf/f;albcre+, p53f/f;albcre+ and Rbf/f; p53f/f;albcre+ mice). Genotype, histologic, immunohistochemical, microarray, quantitative polymerase chain reaction, immunoblot, and comparative genomic hybridization analyses were performed using normal and tumor samples. Comparative microarray analyses were performed against publicly available human microarray data sets. Deletion of RB and p53 from livers of mice deregulated the transcriptional programs associated with human disease. These changes were not sufficient for spontaneous tumorigenesis; potent quiescence mechanisms compensated for loss of these tumor suppressors. In response to hepatocarcinogen-induced damage, distinct and cooperative roles of RB and p53 were revealed; their loss affected cell cycle control, checkpoint response, and genome stability. In damaged tissue, combined loss of RB and p53 resulted in early lesion formation, aggressive tumor progression, and gene expression signatures and histologic characteristics of advanced human hepatocellular carcinoma. The effects RB and p53 loss are determined by the tissue environment; cell stresses that promote aggressive disease reveal the functions of these tumor suppressors. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion

Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.ope

Mitochondrial ROS modulation by ClpXP.

<p>(<b>A</b> and <b>B</b>) PC3 cells transfected with control siRNA (Ctrl) or ClpX- or ClpP-directed siRNA were labeled with CellROX Green Reagent by flow cytometry (<b>A</b>), and staining intensity was quantified (<b>B</b>). H₂O₂ was a control oxidative stimulus. **, <i>p</i> = 0.004; ***, <i>p</i> < 0.0001. (<b>C</b>) The indicated prostate cancer cell types were analyzed for total ROS production as in (<b>A</b>). *, <i>p</i> = 0.03–0.04; **, <i>p</i> = 0.001; ***, <i>p</i> = 0.0008–<0.0001. (<b>D</b>) PC3 cells transfected with the indicated siRNAs as in (<b>A</b>) were analyzed for MitoSOX red mitochondrial superoxide reactivity by fluorescence microscopy and quantified. H₂O₂ was a control oxidative stimulus. **, <i>p</i> = 0.001; ***, <i>p</i> = 0.0004–0.0001. (<b>E</b> and <b>F</b>) PC3 cells stably transduced with control pLKO or shRNA to ClpX or ClpP were analyzed for mitochondrial superoxide production by fluorescence microscopy (<b>E</b>), and staining intensity was quantified (<b>F</b>). ***, <i>p</i> = 0.0004–0.0001. (<b>G</b>) PC3 cells stably transduced with pLKO or ClpX- or ClpP-directed shRNA were analyzed by western blotting. (<b>H</b>) PC3 cells transfected with control siRNA or ClpX- or ClpP-directed siRNA were incubated with vehicle (Veh) or the combination of antioxidants NAC (N) plus mitochondrial-directed MitoTempo (MT) and analyzed by western blotting in the absence or presence of the autophagic flux inhibitor hydroxychloroquine (HCQ). (<b>I</b>) Densitometric quantification of LC3-II/I ratio in vehicle (Veh) or antioxidant-treated PC3 cells in (<b>H</b>). Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002507#pbio.1002507.s003" target="_blank">S3 Data</a>.</p