Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin‐2 (Mfn‐2), which promotes fusion, and dynamin‐related protein (Drp‐1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp‐1/Mfn‐2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp‐1 and decrease in Mfn‐2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn‐2, Drp‐1 inhibition, or Drp‐1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp‐1 inhibition ( P < 0.001). In a xenotransplantation model, Mfn‐2 gene therapy or Drp‐1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm ³ ( P <0.05) with Mfn‐2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm ³ ( P <0.01) with therapeutic Drp‐1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.—Rehman, J., Zhang, H. J., Toth, P. T., Zhang, Y., Marsboom, G., Hong, Z., Salgia, R., Husain, A. N., Wietholt, C., Archer, S. L. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB J. 26, 2175‐2186 (2012). www.fasebj.org

Rationale:: Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by pulmonary vascular obstruction caused, in part, by pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Mitochondrial fragmentation and normoxic activation of hypoxia-inducible factor-1α (HIF-1α) have been observed in PAH PASMCs; however, their relationship and relevance to the development of PAH are unknown. Dynamin-related protein-1 (DRP1) is a GTPase that, when activated by kinases that phosphorylate serine 616, causes mitochondrial fission. It is, however, unknown whether mitochondrial fission is a prerequisite for proliferation. Objective:: We hypothesize that DRP1 activation is responsible for increased mitochondrial fission in PAH PASMCs and that DRP1 inhibition may slow proliferation and have therapeutic potential. Methods and Results:: Experiments were conducted using human control and PAH lungs (n=5) and PASMCs in culture. Parallel experiments were performed in rat lung sections and PASMCs and in rodent PAH models induced by the HIF-1α activator, cobalt, chronic hypoxia, and monocrotaline. HIF-1α activation in human PAH leads to mitochondrial fission by cyclin B1/CDK1–dependent phosphorylation of DRP1 at serine 616. In normal PASMCs, HIF-1α activation by CoCl ₂ or desferrioxamine causes DRP1-mediated fission. HIF-1α inhibition reduces DRP1 activation, prevents fission, and reduces PASMC proliferation. Both the DRP1 inhibitor Mdivi-1 and siDRP1 prevent mitotic fission and arrest PAH PASMCs at the G2/M interphase. Mdivi-1 is antiproliferative in human PAH PASMCs and in rodent models. Mdivi-1 improves exercise capacity, right ventricular function, and hemodynamics in experimental PAH. Conclusions:: DRP-1–mediated mitotic fission is a cell-cycle checkpoint that can be therapeutically targeted in hyperproliferative disorders such as PAH.

Background— Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H ₂ O ₂ , is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this are unknown. Methods and Results— SOD2 expression in PASMCs is decreased in PAH patients and FHRs with PAH. FHR PASMCs have higher proliferation and lower apoptosis rates than Sprague-Dawley rat PASMCs. Moreover, FHR PASMCs have hyperpolarized mitochondria, low H ₂ O ₂ production, and reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing mitochondria, decreasing H ₂ O ₂ , and inhibiting caspase activity. Conversely, SOD2 overexpression in FHR PASMCs or therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PAs versus aortic SMCs and is reversed by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, restoring both SOD2 expression and the ratio of proliferation to apoptosis. Expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs. Conclusions— Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.

Human mesenchymal stem cells (MSCs) are adult multipotent stem cells which can be isolated from bone marrow, adipose tissue as well as other tissues and have the capacity to differentiate into a variety of mesenchymal cell types such as adipocytes, osteoblasts and chondrocytes. Differentiation of stem cells into mature cell types is guided by growth factors and hormones, but recent studies suggest that metabolic shifts occur during differentiation and can modulate the differentiation process. We therefore investigated mitochondrial biogenesis, mitochondrial respiration and the mitochondrial membrane potential during adipogenic differentiation of human MSCs. In addition, we inhibited mitochondrial function to assess its effects on adipogenic differentiation. Our data show that mitochondrial biogenesis and oxygen consumption increase markedly during adipogenic differentiation, and that reducing mitochondrial respiration by hypoxia or by inhibition of the mitochondrial electron transport chain significantly suppresses adipogenic differentiation. Furthermore, we used a novel approach to suppress mitochondrial activity using a specific siRNA-based knockdown of the mitochondrial transcription factor A (TFAM), which also resulted in an inhibition of adipogenic differentiation. Taken together, our data demonstrates that increased mitochondrial activity is a prerequisite for MSC differentiation into adipocytes. These findings suggest that metabolic modulation of adult stem cells can maintain stem cell pluripotency or direct adult stem cell differentiation.