Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming.

Defects in mitochondrial oxidative phosphorylation complexes, altered bioenergetics and metabolic shift are often seen in cancers. Here we show a role for the dysfunction of the electron transport chain component cytochrome c oxidase (CcO) in cancer progression. We show that genetic silencing of the CcO complex by shRNA expression and loss of CcO activity in multiple cell types from the mouse and human sources resulted in metabolic shift to glycolysis, loss of anchorage-dependent growth and acquired invasive phenotypes. Disruption of the CcO complex caused loss of transmembrane potential and induction of Ca2+/Calcineurin-mediated retrograde signaling. Propagation of this signaling includes activation of PI3-kinase, IGF1R and Akt, Ca2(+)-sensitive transcription factors and also TGFß1, MMP16 and periostin, which are involved in oncogenic progression. Whole-genome expression analysis showed the upregulation of genes involved in cell signaling, extracellular matrix interactions, cell morphogenesis, cell motility and migration. The transcription profiles reveal extensive similarity to retrograde signaling initiated by partial mitochondrial DNA depletion, although distinct differences are observed in signaling induced by CcO dysfunction. The possible CcO dysfunction as a biomarker for cancer progression was supported by data showing that esophageal tumors from human patients show reduced CcO subunits IVi1 and Vb in regions that were previously shown to be the hypoxic core of the tumors. Our results show that mitochondrial electron transport chain defect initiates a retrograde signaling. These results suggest that a defect in the CcO complex can potentially induce tumor progression.

Mitochondrial retrograde signaling induces epithelial-mesenchymal transition and generates breast cancer stem cells.

Metastatic breast tumors undergo epithelial-to-mesenchymal transition (EMT), which renders them resistant to therapies targeted to the primary cancers. The mechanistic link between mtDNA (mitochondrial DNA) reduction, often seen in breast cancer patients, and EMT is unknown. We demonstrate that reducing mtDNA content in human mammary epithelial cells (hMECs) activates Calcineurin (Cn)-dependent mitochondrial retrograde signaling pathway, which induces EMT-like reprogramming to fibroblastic morphology, loss of cell polarity, contact inhibition and acquired migratory and invasive phenotype. Notably, mtDNA reduction generates breast cancer stem cells. In addition to retrograde signaling markers, there is an induction of mesenchymal genes but loss of epithelial markers in these cells. The changes are reversed by either restoring the mtDNA content or knockdown of CnAα mRNA, indicating the causal role of retrograde signaling in EMT. Our results point to a new therapeutic strategy for metastatic breast cancers targeted to the mitochondrial retrograde signaling pathway for abrogating EMT and attenuating cancer stem cells, which evade conventional therapies. We report a novel regulatory mechanism by which low mtDNA content generates EMT and cancer stem cells in hMECs.

Growth cones are not required for initial establishment of polarity or differential axon branch growth in cultured hippocampal neurons.

Hippocampal neurons developing in culture exhibit two types of differential, seemingly competitive, process outgrowth in the absence of external cues. During the initial acquisition of polarity, one of several equivalent undifferentiated minor neurites preferentially grows to become the axon. Once the axon has formed, it typically branches, and the branches grow differentially rather than concurrently. In axons with only two branches, growth alternates between branches. In both axon establishment and branch growth alternation, growth among sibling processes or branches must be differentially regulated. We found that elaborate and dynamic growth cones were associated with growth, whereas diminished growth cones were associated with nongrowing processes or branches. To test whether growth cones were necessary for differential growth, growth cone motility was eliminated by application of cytochalasin E. Although cytochalasin treatment before axon formation yielded longer processes overall, a similar percentage of both treated and untreated neurons had one process that grew more rapidly and became much longer than its sibling processes. Immunostaining to visualize dephospho-tau, an axonal marker, demonstrated that these single dominant processes were axons. Axons that formed in cytochalasin were thicker and showed more intense anti-tubulin staining than their sibling processes. Branched axons deprived of growth cones retained a pattern of differential growth and often included alternation. These results indicate that neither formation of a single axon nor differential growth of branches are dependent on growth cone motility and suggest that the neuron can regulate neurite elongation at sites other than at the growth cone.

Role of moving growth cone-like "wave" structures in the outgrowth of cultured hippocampal axons and dendrites.

Hippocampal neurons exhibit periodically recurring growth cone-like structures, referred to as "waves," that emerge at the base of neurites and travel distally to the tip. As a wave nears the tip, the neurite undergoes retraction, and when it reaches the tip, the neurite undergoes a burst of growth. At 1 day in culture, during early axon outgrowth, axons undergo an average 7.5-microm retraction immediately preceding wave arrival at the tip followed by 12-microm growth immediately after arrival (an average net growth of 4.5 microm). In branched axons, waves often selectively travel down one branch or the other. Growth selectively occurs in the branch chosen by the wave. In dendrites, which grow much slower on average, wave-associated retractions are much greater, resulting in less net growth. In the presence of Brefeldin A, which disrupts membrane traffic through the Golgi apparatus and leads to retraction of the axon, axonal waves continue to be associated with both growth spurts and retractions. The magnitude of the growth spurts is not significantly different from untreated axons, but wave-associated retractions are significantly increased. The close association between waves and cyclical elongation suggests that waves may act to bring about this pattern of growth. Our results also show that modulation of regularly occurring retraction phases plays a prominent role in determining average outgrowth rates.

Actin-dependent anterograde movement of growth-cone-like structures along growing hippocampal axons: a novel form of axonal transport?

In time-lapse video recordings of hippocampal neurons in culture, we have identified previously uncharacterized structures, nicknamed "waves," that exhibit lamellipodial activity closely resembling that of growth cones, but which periodically emerge at the base of axons and travel distally at an average rate of 3 microm/min. In electron micrographs of identified waves, the cortical region of the axon appears expanded to either side, forming lamellipodia like those at growth cones. No other gross differences were noted in the ultrastructural features of the axon shaft at the site of a wave. Immunocytochemistry revealed that waves contain a marked concentration of F-actin, GAP-43, cortactin, and ezrin or a related protein, constituents that are also concentrated in growth cones. Treatment with the actin-disrupting agent cytochalasin B caused a reversible collapse of lamellipodia and cessation of the forward movement of individual waves along the axon, indicating that their anterograde transport is dependent on intact actin filaments. Treatment with the microtubule-depolymerizing agent nocodazole led to a rapid disorganization of wave structure and a subsequent suppression of wave activity that may reflect a role of microtubules in actin organization. The results suggest that actin and other cytoskeletal components concentrated in growth cones may be transported together as growth-cone-like structures from the cell body to the axon tip via an actin-dependent mechanism.

Bergmann glia require continuous association with Purkinje cells for normal phenotype expression.

Bergmann glia (Bg) respond to the early postnatal Purkinje cell (Pc) death in Lurcher (Lc) mutant mouse cerebellum by down-regulating expression of the enzyme glycerol-3-phosphate dehydrogenase (GPDH). To determine whether glial GPDH expression requires the continued presence of Pcs in adults, we used single intracerebellar injections of kainic acid to kill Pcs in wild-type mice from 7 weeks to 11 months old. Bg at all ages tested responded to Pc loss by down-regulating GPDH expression. To learn whether a high level of GPDH could be reinduced following down-regulation in Lc Bg, we grafted wild-type fetal Pcs into Lc cerebella. The influence of grafted Pcs on GPDH expression is host-age and implant-position dependent. Only Pcs implanted into hosts less than 6 weeks old were later found to be associated with GPDH-positive Bg. Grafted Pcs that migrated into the anterior folia of young hosts were more likely to be associated with GPDH-positive Bg than Pcs migrating to other positions. EM analysis showed that Bg ensheathment of grafted Pcs is thinner and more discontinuous, but qualitatively similar to normal. The results suggest that the interaction between host Bg and grafted Pcs can sustain elevated GPDH expression in Bg that have not yet down-regulated, but is not adequate to reinduce expression in those cells that have.