Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression.

Mitochondria are signaling organelles implicated in cancer, but the mechanisms are elusive. Here, we show that Parkin, an E3 ubiquitination (Ub) ligase altered in Parkinson's disease, forms a complex with the regulator of cell motility, Kindlin-2 (K2), at mitochondria of tumor cells. In turn, Parkin ubiquitinates Lys581 and Lys582 using Lys48 linkages, resulting in proteasomal degradation of K2 and shortened half-life from ∼5 h to ∼1.5 h. Loss of K2 inhibits focal adhesion turnover and ß1 integrin activation, impairs membrane lamellipodia size and frequency, and inhibits mitochondrial dynamics, altogether suppressing tumor cell-extracellular matrix interactions, migration, and invasion. Conversely, Parkin does not affect tumor cell proliferation, cell cycle transitions, or apoptosis. Expression of a Parkin Ub-resistant K2 Lys581Ala/Lys582Ala double mutant is sufficient to restore membrane lamellipodia dynamics, correct mitochondrial fusion/fission, and preserve single-cell migration and invasion. In a 3D model of mammary gland developmental morphogenesis, impaired K2 Ub drives multiple oncogenic traits of EMT, increased cell proliferation, reduced apoptosis, and disrupted basal-apical polarity. Therefore, deregulated K2 is a potent oncogene, and its Ub by Parkin enables mitochondria-associated metastasis suppression.

Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth.

The role of mitochondria in cancer continues to be debated, and whether exploitation of mitochondrial functions is a general hallmark of malignancy or a tumor- or context-specific response is still unknown. Using a variety of cancer cell lines and several technical approaches, including siRNA-mediated gene silencing, ChIP assays, global metabolomics and focused metabolite analyses, bioenergetics, and cell viability assays, we show that two oncogenic Myc proteins, c-Myc and N-Myc, transcriptionally control the expression of the mitochondrial chaperone TNFR-associated protein-1 (TRAP1) in cancer. In turn, this Myc-mediated regulation preserved the folding and function of mitochondrial oxidative phosphorylation (OXPHOS) complex II and IV subunits, dampened reactive oxygen species production, and enabled oxidative bioenergetics in tumor cells. Of note, we found that genetic or pharmacological targeting of this pathway shuts off tumor cell motility and invasion, kills Myc-expressing cells in a TRAP1-dependent manner, and suppresses primary and metastatic tumor growth in vivo We conclude that exploitation of mitochondrial functions is a general trait of tumorigenesis and that this reliance of cancer cells on mitochondrial OXPHOS pathways could offer an actionable therapeutic target in the clinic.

IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α-regulated pseudohypoxic state.

The role of mitochondria in cancer continues to be debated and paradoxically implicated in opposing functions in tumor growth and tumor suppression. To understand this dichotomy, we explored the function of mitochondrial isocitrate dehydrogenase (IDH)2, a tricarboxylic acid cycle enzyme mutated in subsets of acute leukemias and gliomas, in cancer. Silencing of IDH2 in prostate cancer cells impaired oxidative bioenergetics, elevated reactive oxygen species (ROS) production, and promoted exaggerated mitochondrial dynamics. This was associated with increased subcellular mitochondrial trafficking, turnover of membrane focal adhesion complexes, and enhanced tumor cell migration and invasion, without changes in cell cycle progression. Mechanistically, loss of IDH2 caused ROS-dependent stabilization of hypoxia-inducible factor-1α in normoxia, which was required for increased mitochondrial trafficking and tumor cell movements. Therefore, IDH2 is a dual regulator of cancer bioenergetics and tumor cell motility. This pathway may reprogram mitochondrial dynamics to differentially adjust energy production or promote tumor cell invasion in response to microenvironment conditions.-Wang, Y., Agarwal, E., Bertolini, I., Ghosh, J. C., Seo, J. H., Altieri, D. C. IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α-regulated pseudohypoxic state.

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.

Cyclophilin D extramitochondrial signaling controls cell cycle progression and chemokine-directed cell motility.

Mitochondria control bioenergetics and cell fate decisions, but how they influence nuclear gene expression is understood poorly. Here, we show that deletion or reduction in the levels of cyclophilin D (CypD, also called Ppif), a mitochondrial matrix peptidyl prolyl isomerase and apoptosis regulator, results in increased cell proliferation and enhanced cell migration and invasion. These responses are associated with extensive transcriptional changes, modulation of a chemokine/chemokine receptor gene signature, and activation of the pleiotropic inflammatory mediator, STAT3. In the absence of CypD, active STAT3 enhances cell proliferation via accelerated entry into S-phase and stimulates autocrine/paracrine cell motility through Cxcl12-Cxcr4-directed chemotaxis. Therefore, CypD directs mitochondria-to-nuclei inflammatory gene expression in normal and tumor cells. This pathway may contribute to malignant traits under conditions of CypD modulation.

Intercellular hif1α reprograms mammary progenitors and myeloid immune evasion to drive high-risk breast lesions.

The origin of breast cancer, whether primary or recurrent, is unknown. Here, we show that invasive breast cancer cells exposed to hypoxia release small extracellular vesicles (sEVs) that disrupt the differentiation of normal mammary epithelia, expand stem and luminal progenitor cells, and induce atypical ductal hyperplasia and intraepithelial neoplasia. This was accompanied by systemic immunosuppression with increased myeloid cell release of the alarmin S100A9 and oncogenic traits of epithelial-mesenchymal transition, angiogenesis, and local and disseminated luminal cell invasion in vivo. In the presence of a mammary gland driver oncogene (MMTV-PyMT), hypoxic sEVs accelerated bilateral breast cancer onset and progression. Mechanistically, genetic or pharmacologic targeting of hypoxia-inducible factor-1α (HIF1α) packaged in hypoxic sEVs or homozygous deletion of S100A9 normalized mammary gland differentiation, restored T cell function, and prevented atypical hyperplasia. The transcriptome of sEV-induced mammary gland lesions resembled luminal breast cancer, and detection of HIF1α in plasma circulating sEVs from luminal breast cancer patients correlated with disease recurrence. Therefore, sEV-HIF1α signaling drives both local and systemic mechanisms of mammary gland transformation at high risk for evolution to multifocal breast cancer. This pathway may provide a readily accessible biomarker of luminal breast cancer progression.

NFκB activation by hypoxic small extracellular vesicles drives oncogenic reprogramming in a breast cancer microenvironment.

Small extracellular vesicles (sEV) contribute to the crosstalk between tumor cells and stroma, but the underlying signals are elusive. Here, we show that sEV generated by breast cancer cells in hypoxic (sEVHYP), but not normoxic (sEVNORM) conditions activate NFκB in recipient normal mammary epithelial cells. This increases the production and release of inflammatory cytokines, promotes mitochondrial dynamics leading to heightened cell motility and disrupts 3D mammary acini architecture with aberrant cell proliferation, reduced apoptosis and EMT. Mechanistically, Integrin-Linked Kinase packaged in sEVHYP via HIF1α is sufficient to activate NFκB in the normal mammary epithelium, in vivo. Therefore, sEVHYP activation of NFκB drives multiple oncogenic steps of inflammation, mitochondrial dynamics, and mammary gland morphogenesis in a breast cancer microenvironment.

Mitochondrial fitness and cancer risk.

Changes in metabolism are a hallmark of cancer, but molecular signatures of altered bioenergetics to aid in clinical decision-making do not currently exist. We recently identified a group of human tumors with constitutively reduced expression of the mitochondrial structural protein, Mic60, also called mitofilin or inner membrane mitochondrial protein (IMMT). These Mic60-low tumors exhibit severe loss of mitochondrial fitness, paradoxically accompanied by increased metastatic propensity and upregulation of a unique transcriptome of Interferon (IFN) signaling and Senescence-Associated Secretory Phenotype (SASP). Here, we show that an optimized, 11-gene signature of Mic60-low tumors is differentially expressed in multiple malignancies, compared to normal tissues, and correlates with poor patient outcome. When analyzed in three independent patient cohorts of pancreatic ductal adenocarcinoma (PDAC), the Mic60-low gene signature was associated with aggressive disease variants, local inflammation, FOLFIRINOX failure and shortened survival, independently of age, gender, or stage. Therefore, the 11-gene Mic60-low signature may provide an easily accessible molecular tool to stratify patient risk in PDAC and potentially other malignancies.

A cancer ubiquitome landscape identifies metabolic reprogramming as target of Parkin tumor suppression.

Changes in metabolism that affect mitochondrial and glycolytic networks are hallmarks of cancer, but their impact in disease is still elusive. Using global proteomics and ubiquitome screens, we now show that Parkin, an E3 ubiquitin ligase and key effector of mitophagy altered in Parkinson's disease, shuts off mitochondrial dynamics and inhibits the non-oxidative phase of the pentose phosphate pathway. This blocks tumor cell movements, creates metabolic and oxidative stress, and inhibits primary and metastatic tumor growth. Uniformly down-regulated in cancer patients, Parkin tumor suppression requires its E3 ligase function, is reversed by antioxidants, and is independent of mitophagy. These data demonstrate that cancer metabolic networks are potent oncogenes directly targeted by endogenous tumor suppression.

Small Extracellular Vesicle Regulation of Mitochondrial Dynamics Reprograms a Hypoxic Tumor Microenvironment.

The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium.

The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells.

Mitochondria are signaling hubs in eukaryotic cells. Here, we showed that the mitochondrial FUN14 domain-containing protein-1 (FUNDC1), an effector of Parkin-independent mitophagy, also participates in cellular plasticity by sustaining oxidative bioenergetics, buffering ROS production, and supporting cell proliferation. Targeting this pathway in cancer cells suppressed tumor growth but rendered transformed cells more motile and invasive in a manner dependent on ROS-mediated mitochondrial dynamics and mitochondrial repositioning to the cortical cytoskeleton. Global metabolomics and proteomics profiling identified a FUNDC1 interactome at the mitochondrial inner membrane, comprising the AAA+ protease, LonP1, and subunits of oxidative phosphorylation, complex V (ATP synthase). Independently of its previously identified role in mitophagy, FUNDC1 enabled LonP1 proteostasis, which in turn preserved complex V function and decreased ROS generation. Therefore, mitochondrial reprogramming by a FUNDC1-LonP1 axis controls tumor cell plasticity by switching between proliferative and invasive states in cancer.

Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits.

Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. Here we show that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.

Myc Regulation of a Mitochondrial Trafficking Network Mediates Tumor Cell Invasion and Metastasis.

The Myc gene is a universal oncogene that promotes aggressive cancer, but its role in metastasis has remained elusive. Here, we show that Myc transcriptionally controls a gene network of subcellular mitochondrial trafficking that includes the atypical mitochondrial GTPases RHOT1 and RHOT2, the adapter protein TRAK2, the anterograde motor Kif5B, and an effector of mitochondrial fission, Drp1. Interference with this pathway deregulates mitochondrial dynamics, shuts off subcellular organelle movements, and prevents the recruitment of mitochondria to the cortical cytoskeleton of tumor cells. In turn, this inhibits tumor chemotaxis, blocks cell invasion, and prevents metastatic spreading in preclinical models. Therefore, Myc regulation of mitochondrial trafficking enables tumor cell motility and metastasis.

Activated checkpoint kinase 2 provides a survival signal for tumor cells.

Tumor cells often become resistant to DNA damage-based therapy; however, the underlying mechanisms are not yet understood. Here, we show that tumor cells exposed to DNA damage counteract cell death by releasing the antiapoptotic protein, survivin, from mitochondria. This is independent of p53, and requires activated checkpoint kinase 2 (Chk2), a putative tumor suppressor. Molecular or genetic targeting of Chk2 prevents the release of survivin from mitochondria, enhances DNA damage-induced tumor cell apoptosis, and inhibits the growth of resistant in vivo tumors. Therefore, activated Chk2 circumvents its own tumor-suppressive functions by promoting tumor cell survival. Inhibiting Chk2 in combination with DNA-damaging agents may provide a rational approach for treating resistant tumors.

Chk2 phosphorylation of BRCA1 regulates DNA double-strand break repair.

The pathway determining malignant cellular transformation, which depends upon mutation of the BRCA1 tumor suppressor gene, is poorly defined. A growing body of evidence suggests that promotion of DNA double-strand break repair by homologous recombination (HR) may be the means by which BRCA1 maintains genomic stability, while a role of BRCA1 in error-prone nonhomologous recombination (NHR) processes has just begun to be elucidated. The BRCA1 protein becomes phosphorylated in response to DNA damage, but the effects of phosphorylation on recombinational repair are unknown. In this study, we tested the hypothesis that the BRCA1-mediated regulation of recombination requires the Chk2- and ATM-dependent phosphorylation sites. We studied Rad51-dependent HR and random chromosomal integration of linearized plasmid DNA, a subtype of NHR, which we demonstrate to be dependent on the Mre11-Rad50-Nbs1 complex. Prevention of Chk2-mediated phosphorylation via mutation of the serine 988 residue of BRCA1 disrupted both the BRCA1-dependent promotion of HR and the suppression of NHR. Similar results were obtained when endogenous Chk2 kinase activity was inhibited by expression of a dominant-negative Chk2 mutant. Surprisingly, the opposing regulation of HR and NHR did not require the ATM phosphorylation sites on serines 1423 and 1524. Together, these data suggest a functional link between recombination control and breast cancer predisposition in carriers of Chk2 and BRCA1 germ line mutations. We propose a dual regulatory role for BRCA1 in maintaining genome integrity, whereby BRCA1 phosphorylation status controls the selectivity of repair events dictated by HR and error-prone NHR.

Activation of p53-dependent apoptosis by acute ablation of glycogen synthase kinase-3beta in colorectal cancer cells.

PURPOSE: The restoration of checkpoint mechanisms may provide a rational anticancer approach, but the molecular circuitries of how this can be achieved therapeutically are poorly understood. A pivotal signaling network in colorectal cancer cells involves glycogen synthase kinase-3beta (GSK3beta), a multifunctional kinase whose role in tumor cell survival is not defined. EXPERIMENTAL DESIGN: We used molecular, genetic, and pharmacologic antagonists of GSK3beta in p53+/+ or p53-/- colorectal cancer cells. We monitored kinase activity in immunoprecipitation, protein expression by immunoblotting, and cell death by multiparametric flow cytometry. A xenograft colorectal cancer model was used to study antitumor activity in vivo. RESULTS: Treatment of p53+/+ colorectal cancer cells with pharmacologic inhibitors of GSK3beta resulted in sustained elevation of p53, with up-regulation of p21(Waf1/Cip1) and loss of survivin levels. Molecular targeting of GSK3beta by overexpression of a GSK3beta dominant-negative mutant, or acute-silencing of GSK3beta by RNA interference, reproduced the induction of transcriptionally active p53 in colorectal cancer cells. This pathway was recapitulated by deregulated Wnt/T-cell factor signaling, with elevation of the tumor suppressor p14ARF, and reduced expression of the p53 antagonist, MDM2. Rather than cell cycle arrest, GSK3beta blockade resulted in p53-dependent apoptosis, which was contributed by acute loss of survivin and inhibition of colorectal cancer growth in mice. CONCLUSIONS: Acute ablation of GSK3beta in colorectal cancer cells activates p53-dependent apoptosis and antagonizes tumor growth. This pathway may be exploited for rational treatment of colorectal cancer patients retaining wild-type p53.

Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.