Unchecked oxidative stress in skeletal muscle prevents outgrowth of disseminated tumour cells.

Skeletal muscle has long been recognized as an inhospitable site for disseminated tumour cells (DTCs). Yet its antimetastatic nature has eluded a thorough mechanistic examination. Here, we show that DTCs traffic to and persist within skeletal muscle in mice and in humans, which raises the question of how this tissue suppresses colonization. Results from mouse and organotypic culture models along with metabolomic profiling suggested that skeletal muscle imposes a sustained oxidative stress on DTCs that impairs their proliferation. Functional studies demonstrated that disrupting reduction-oxidation homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in a more fertile organ: the lung. Conversely, enhancement of the antioxidant potential of tumour cells through ectopic expression of catalase in the tumour or host mitochondria allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. A thorough understanding of this biology could reveal previously undocumented DTC vulnerabilities that can be exploited to prevent metastasis in other more susceptible tissues.

Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumor cell (DTC) dormancy in this organ is limited. Here using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micrometastatic state as the rate-limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein, thereby sequestering it from the nucleus and preventing its prometastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.

The PI3K/mTOR inhibitor Gedatolisib eliminates dormant breast cancer cells in organotypic culture, but fails to prevent metastasis in preclinical settings.

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin-ß1-mediated interactions. Because integrin-ß1-targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin-ß1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin-ß1, we determined that PI3K inhibition with either a tool compounds or a compound (PF-05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib-with or without genotoxic therapy-would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple-negative or estrogen receptor-positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle-treated animals) or in combination with dose-dense doxorubicin and cyclophosphamide (vs. animals treated only with dose-dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin-ß1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Targeting the perivascular niche sensitizes disseminated tumour cells to chemotherapy.

The presence of disseminated tumour cells (DTCs) in bone marrow is predictive of poor metastasis-free survival of patients with breast cancer with localized disease. DTCs persist in distant tissues despite systemic administration of adjuvant chemotherapy. Many assume that this is because the majority of DTCs are quiescent. Here, we challenge this notion and provide evidence that the microenvironment of DTCs protects them from chemotherapy, independent of cell cycle status. We show that chemoresistant DTCs occupy the perivascular niche (PVN) of distant tissues, where they are protected from therapy by vascular endothelium. Inhibiting integrin-mediated interactions between DTCs and the PVN, driven partly by endothelial-derived von Willebrand factor and vascular cell adhesion molecule 1, sensitizes DTCs to chemotherapy. Importantly, chemosensitization is achieved without inducing DTC proliferation or exacerbating chemotherapy-associated toxicities, and ultimately results in prevention of bone metastasis. This suggests that prefacing adjuvant therapy with integrin inhibitors is a viable clinical strategy to eradicate DTCs and prevent metastasis.

Reprogramming of the Tumor in the Hypoxic Niche: The Emerging Concept and Associated Therapeutic Strategies.

Hypoxia exerts a profound impact on diverse aspects of cancer biology. Increasing evidence has revealed novel functions of hypoxia in cancer cell epigenomics, epitranscriptomics, metabolism, and intercellular communication, all hotspots of cancer research. Several drugs have been developed to target intratumoral hypoxia and have entered clinical trials to treat refractory tumors. However, direct targeting of hypoxia signaling still has limitations in the clinic with regard to cancer progression and resistance to therapy. Comprehensive understanding of the molecular mechanisms by which hypoxia reshapes tumors and their microenvironment, as well as how tumor cells adapt to and thrive in hypoxic conditions, will therefore continue to be a focus of cancer research and will provide new directions for hypoxic tumor treatment.

Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future.

Although brain tumors have been known tremendously over the past decade, there are still many problems to be solved. The etiology of brain tumors is not well understood and the treatment remains modest. There is in great need to develop a suitable brain tumor models that faithfully mirror the etiology of human brain neoplasm and subsequently get more efficient therapeutic approaches for these disorders. In this review, we described the current status of animal models of brain tumors and analyzed their advantages and disadvantages. Additionally, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a versatile genome editing technology for investigating the functions of target genes, and its application were also introduced in our present work. We firstly proposed that brain tumor modeling could be well established via CRISPR/Cas9 techniques. And CRISPR/Cas9-mediated brain tumor modeling was likely to be more suitable for figuring out the pathogenesis of brain tumors, as CRISPR/Cas9 platform was a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that were closely linked with brain tumors.

Olig1 Acetylation and Nuclear Export Mediate Oligodendrocyte Development.

The oligodendrocyte transcription factor Olig1 is critical for both oligodendrocyte development and remyelination in mice. Nuclear to cytoplasmic translocation of Olig1 protein occurs during brain development and in multiple sclerosis, but the detailed molecular mechanism of this translocation remains elusive. Here, we report that Olig1 acetylation and deacetylation drive its active translocation between the nucleus and the cytoplasm in both mouse and rat oligodendrocytes. We identified three functional nuclear export sequences (NES) localized in the basic helix-loop-helix domain and one specific acetylation site at Lys 150 (human Olig1) in NES1. Olig1 acetylation and deacetylation are regulated by the acetyltransferase CREB-binding protein and the histone deacetylases HDAC1, HDAC3, and HDAC10. Acetylation of Olig1 decreased its chromatin association, increased its interaction with inhibitor of DNA binding 2 and facilitated its retention in the cytoplasm of mature oligodendrocytes. These studies establish that acetylation of Olig1 regulates its chromatin dissociation and subsequent translocation to the cytoplasm and is required for its function in oligodendrocyte maturation.

A Novel Function of TET2 in CNS: Sustaining Neuronal Survival.

DNA dioxygenases Ten-Eleven Translocation (TET) proteins can catalyze the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), and thereby alter the epigenetic state of DNA. The TET family includes TET1, TET2 and TET3 members in mammals. Recently, accumulative research uncovered that TET1-3 occur abundantly in the central nervous system (CNS), and their biological functions have just begun to be investigated. In the present study, we demonstrated that mRNA and protein of TET2 were highly expressed in the cerebral cortex and hippocampus along the whole brain-development process. Further studies showed that TET2 was expressed in various types of cells, especially in most neurons. Subcellular distribution pattern implicated that TET2 is localized in both nucleus and cytoplasm of neurons. Down-regulation of TET2 in cultured cortical neurons with RNA interference implied that TET2 was required for cell survival. In all, our results indicate that neuronal TET2 is positively involved in the regulation of cell survival.

Olig1 function is required for oligodendrocyte differentiation in the mouse brain.

Oligodendrocyte differentiation and myelination are tightly regulated processes orchestrated by a complex transcriptional network. Two bHLH transcription factors in this network, Olig1 and Olig2, are expressed exclusively by oligodendrocytes after late embryonic development. Although the role of Olig2 in the lineage is well established, the role of Olig1 is still unclear. The current studies analyzed the function of Olig1 in oligodendrocyte differentiation and developmental myelination in brain. Both oligodendrocyte progenitor cell commitment and oligodendrocyte differentiation were impaired in the corpus callosum of Olig1-null mice, resulting in hypomyelination throughout adulthood in the brain. As seen in previous studies with this mouse line, although there was an early myelination deficit in the spinal cord, essentially full recovery with normal spinal cord myelination was seen. Intriguingly, this regional difference may be partially attributed to compensatory upregulation of Olig2 protein expression in the spinal cord after Olig1 deletion, which is not seen in brain. The current study demonstrates a unique role for Olig1 in promoting oligodendrocyte progenitor cell commitment, differentiation, and subsequent myelination primarily in brain, but not spinal cord.

Interaction of mTOR and Erk1/2 signaling to regulate oligodendrocyte differentiation.

A multitude of factors regulate oligodendrocyte differentiation and remyelination, and to elucidate the mechanisms underlying this process, we analyzed the interactions of known signaling pathways involved in these processes. Previous work from our lab and others shows that Akt, mTOR, and Erk 1/2 are major signaling pathways regulating oligodendrocyte differentiation and myelination in vitro and in vivo. However, the relative contribution of the different pathways has been difficult to establish because the impact of inhibiting one pathway in in vitro cell culture models or in vivo may alter signaling through the other pathway. These studies were undertaken to clarify the interactions between these major pathways and understand more specifically the crosstalk between them. Oligodendrocyte differentiation in vitro required Akt, mTOR, and Erk 1/2 signaling, as inhibition of Akt, mTOR, or Erk 1/2 resulted in a significant decrease of myelin basic protein mRNA and protein expression. Interestingly, while inhibition of the Erk1/2 pathway had little impact on Akt/mTOR signaling, inhibition of the Akt/mTOR pathways significantly increased Erk1/2 signaling, although not enough to overcome the loss of Akt/mTOR signaling in the regulation of oligodendrocyte differentiation. Furthermore, such crosstalk was also noted in an in vivo context, after mTOR inhibition by rapamycin treatment of perinatal pups. GLIA 2014;62:2096-2109.

Conditional ablation of raptor or rictor has differential impact on oligodendrocyte differentiation and CNS myelination.

During CNS development, oligodendrocytes, the myelinating glia of the CNS, progress through multiple transitory stages before terminating into fully mature cells. Oligodendrocyte differentiation and myelination is a tightly regulated process requiring extracellular signals to converge to elicit specific translational and transcriptional changes. Our lab has previously shown that the protein kinases, Akt and mammalian Target of Rapamycin (mTOR), are important regulators of CNS myelination in vivo. mTOR functions through two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2, by binding to either Raptor or Rictor, respectively. To establish whether the impact of mTOR on CNS myelination results from unique functions of mTORC1 or mTORC2 during CNS myelination, we conditionally ablated either Raptor or Rictor in the oligodendrocyte lineage, in vivo. We show that Raptor (mTORC1) is a positive regulator of developmental CNS mouse myelination when mTORC2 is functional, whereas Rictor (mTORC2) ablation has a modest positive effect on oligodendrocyte differentiation, and very little effect on myelination, when mTORC1 is functional. Also, we show that loss of Raptor in oligodendrocytes results in differential dysmyelination in specific areas of the CNS, with the greatest impact on spinal cord myelination.

Dynamics of ten-eleven translocation hydroxylase family proteins and 5-hydroxymethylcytosine in oligodendrocyte differentiation.

The ten-eleven translocation (TET) family of methylcytosine dioxygenases catalyze oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and promote DNA demethylation. Despite the abundance of 5hmC and TET proteins in the brain, little is known about their role in oligodendrocytes (OLs). Here, we analyzed TET expression during OL development in vivo and in vitro, and found that three TET family members possess unique subcellular and temporal expression patterns. Furthermore, the level of 5hmC exhibits dynamic changes during OL maturation, which implies that 5hmC modification may play a role in the expression of critical genes necessary for OL maturation. siRNA-mediated silencing of the TET family proteins in OLs demonstrated that each of the TET proteins is required for OL differentiation. However, based on their unique domain structures, we speculate that the three TET members may function by different mechanisms. In summary, we have established the temporal expression of TET proteins and the dynamic level of 5hmC during OL development and demonstrate that all three TET members are necessary for OL differentiation.

mTOR: a link from the extracellular milieu to transcriptional regulation of oligodendrocyte development.

Oligodendrocyte development is controlled by numerous extracellular signals that regulate a series of transcription factors that promote the differentiation of oligodendrocyte progenitor cells to myelinating cells in the central nervous system. A major element of this regulatory system that has only recently been studied is the intracellular signalling from surface receptors to transcription factors to down-regulate inhibitors and up-regulate inducers of oligodendrocyte differentiation and myelination. The current review focuses on one such pathway: the mTOR (mammalian target of rapamycin) pathway, which integrates signals in many cell systems and induces cell responses including cell proliferation and cell differentiation. This review describes the known functions of mTOR as they relate to oligodendrocyte development, and its recently discovered impact on oligodendrocyte differentiation and myelination. A potential model for its role in oligodendrocyte development is proposed.