The Impact of Centrosome Pathologies on Prostate Cancer Development and Progression.

The significant role of centrosomes in cancer cell proliferation has been well recognized (reviewed in Schatten H, Histochem Cell Biol 129:667-86 (2008); Schatten H, Sun Q-Y, Microsc Microanal 17(4):506-512 (2011); Schatten H, Sun Q-Y, Reprod Fertil Dev. https://doi.org/10.1071/RD14493 (2015a); Schatten H, Sun Q-Y, Centrosome-microtubule interactions in health, disease, and disorders. In: Schatten H (ed) The cytoskeleton in health and disease. Springer Science+Business Media, New York (2015b)) and new research has generated new interest and new insights into centrosomes as potential targets for cancer-specific therapies. The centrosome is a key organelle serving multiple functions through its primary functions as microtubule organizing center (MTOC) that is also an important communication center for processes involved in cellular regulation; transport to and away from centrosome-organized microtubules along microtubules is essential for cellular activities including signal transduction and metabolic activities. New research on cancer cell centrosomes has generated new insights into centrosome dysfunctions in cancer cells in which centrosome phosphorylation, balance of centrosomal proteins, centrosome regulation and duplication are impaired. Among the hallmarks of cancer cells are multipolar spindles or abnormal bipolar spindles that are formed as a result of centrosome protein expression imbalances, abnormalities in centrosome structure and abnormalities in clustering of centrosomal components that are critical for bipolar mitotic apparatus formation. Centrosome abnormalities in cancer cells can be the result of multiple factors including environmental influences and toxicants that can affect centrosome functions by inducing centrosome pathologies leading to abnormal cancer cell proliferation. These topics are addressed in this review with focus on prostate-specific therapy strategies to target centrosome abnormalities. We will also address loss of cell polarity in cancer cells in which centrosome dysfunctions play a role as well as the loss of primary cilia in prostate cancer development and progression.

Mammalian complex I pumps 4 protons per 2 electrons at high and physiological proton motive force in living cells.

Mitochondrial complex I couples electron transfer between matrix NADH and inner-membrane ubiquinone to the pumping of protons against a proton motive force. The accepted proton pumping stoichiometry was 4 protons per 2 electrons transferred (4H(+)/2e(-)) but it has been suggested that stoichiometry may be 3H(+)/2e(-) based on the identification of only 3 proton pumping units in the crystal structure and a revision of the previous experimental data. Measurement of proton pumping stoichiometry is challenging because, even in isolated mitochondria, it is difficult to measure the proton motive force while simultaneously measuring the redox potentials of the NADH/NAD(+) and ubiquinol/ubiquinone pools. Here we employ a new method to quantify the proton motive force in living cells from the redox poise of the bc(1) complex measured using multiwavelength cell spectroscopy and show that the correct stoichiometry for complex I is 4H(+)/2e(-) in mouse and human cells at high and physiological proton motive force.

Acute mitochondrial inhibition by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitors regulates proliferation.

The Ras-MEK1/2-ERK1/2 kinase signaling pathway regulates proliferation, survival, and differentiation and, because it is often aberrant in tumors, is a popular target for small molecule inhibition. A novel metabolic analysis that measures the real-time oxidation state of NAD(H) and the hemes of the electron transport chain and oxygen consumption within intact, living cells found that structurally distinct MEK1/2 inhibitors had an immediate, dose-dependent effect on mitochondrial metabolism. The inhibitors U0126, MIIC and PD98059 caused NAD(H) reduction, heme oxidation, and decreased oxygen consumption, characteristic of complex I inhibition. PD198306, an orally active MEK1/2 inhibitor, acted as an uncoupler. Each MEK1/2 inhibitor depleted phosphorylated ERK1/2 and inhibited proliferation, but the most robust antiproliferative effects always correlated with the metabolic failure which followed mitochondrial inhibition rather than inhibition of MEK1/2. This warrants rethinking the role of ERK1/2 in proliferation and emphasizes the importance of mitochondrial function in this process.

Cytochrome c is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization.

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa(3)) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be approximately 62% oxidized before MOMP and became approximately 70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc(1) with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.

Inhibition of either phosphatidylinositol 3-kinase/Akt or the mitogen/extracellular-regulated kinase, MEK/ERK, signaling pathways suppress growth of breast cancer cell lines, but MEK/ERK signaling is critical for cell survival.

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

Induction of AP-1 activity by androgen activation of the androgen receptor in LNCaP human prostate carcinoma cells.

BACKGROUND: The androgen receptor and activator protein-1 (AP-1) transcription factors affect growth regulation in normal and cancerous prostate cells. Effects of androgen-activated androgen receptor on AP-1 activity were determined in the LNCaP human prostate carcinoma cell model. METHODS: Cells were exposed to 1 nM androgen +/- antiandrogen bicalutamide. Cellular growth and cell cycle effects were determined by DNA, viability, and bromodeoxyuridine (BrdU) fluorescence activated cell sorter (FACS) assays. AP-1 effects were determined by an AP-1-luciferase enzyme reporter vector for transcriptional activity, electrophoretic mobility shift assay (EMSA)/antibody supershift for DNA-binding, quantitative RT-PCR for mRNA, and immunoblot for protein. RESULTS: Androgen induced G(1) growth arrest. This growth arrest was abrogated by treatment with bicalutamide, demonstrating that growth arrest by androgen was due to androgen receptor activation. Concurrently, AP-1 DNA-binding and transcriptional activity was induced over 96 hr androgen exposure, which was also inhibited by bicalutamide. Interestingly, although no change in AP-1 transcriptional activity was observed 24 hr after androgen exposure, there was an increase in Fra-2 expression and AP-1 DNA-binding. Paradoxically, while Fra-2 mRNA and protein levels continued to increase, binding of Fra-2 to the AP-1 site decreased over 96 hr, with a concomitant increase in JunD AP-1-binding and a marked increase in expression of the 35 kDa form of JunD. Enhanced expression of this short form of JunD is a novel effect of androgen exposure that occurred during the 24-96 hr time period, as growth effects emerged. CONCLUSION: Activation of androgen receptor by androgen induces changes in AP-1 activity and AP-1 factor DNA-binding that may contribute significantly to androgen-induced changes in prostate cancer cell growth.