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Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Emerging evidence point to a crucial role for non-coding RNAs in modulating homeostatic signaling under physiological and pathological conditions. MicroRNAs, the best-characterized non-coding RNAs to date, can exquisitely integrate spatial and temporal signals in complex networks, thereby confer specificity and sensitivity to tissue response to changes in the microenvironment. MicroRNAs appear as preferential partners for Receptor Tyrosine Kinases (RTKs) in mediating signaling under stress conditions. Stress signaling can be especially relevant to disease. Here we focus on the ability of microRNAs to mediate RTK signaling in cancer, by acting as both tumor suppressors and oncogenes. We will provide a few general examples of microRNAs modulating specific tumorigenic functions downstream of RTK signaling and integrate oncogenic signals from multiple RTKs. A special focus will be devoted to epidermal growth factor receptor (EGFR) signaling, a system offering relatively rich information. We will explore the role of selected microRNAs as bidirectional modulators of EGFR functions in cancer cells. In addition, we will present the emerging evidence for microRNAs being specifically modulated by oncogenic EGFR mutants and we will discuss how this impinges on EGFRmut driven chemoresistance, which fits into the tumor heterogeneity-driven cancer progression. Finally, we discuss how other non-coding RNA species are emerging as important modulators of cancer progression and why the scenario depicted herein is destined to become increasingly complex in the future.

Metformin is a commonly prescribed type II diabetes medication that exhibits promising anticancer effects. Recently, these effects were found to be associated, at least in part, with a modulation of microRNA expression. However, the mechanisms by which single modulated microRNAs mediate the anticancer effects of metformin are not entirely clear and knowledge of such a process could be vital to maximize the potential therapeutic benefits of this safe and well-tolerated therapy. Our analysis here revealed that the expression of miR-21-5p was downregulated in multiple breast cancer cell lines treated with pharmacologically relevant doses of metformin. Interestingly, the inhibition of miR-21-5p following metformin treatment was also observed in mouse breast cancer xenografts and in sera from 96 breast cancer patients. This modulation occurred at the levels of both pri-miR-21 and pre-miR-21, suggesting transcriptional modulation. Antagomir-mediated ablation of miR-21-5p phenocopied the effects of metformin on both the clonogenicity and migration of the treated cells, while ectopic expression of miR-21-5p had the opposite effect. Mechanistically, this reduction in miR-21-5p enhanced the expression of critical upstream activators of the AMP-activated protein kinase, calcium-binding protein 39-like and Sestrin-1, leading to AMP-activated protein kinase activation and inhibition of mammalian target of rapamycin signaling. Importantly, these effects of metformin were synergistic with those of everolimus, a clinically relevant mammalian target of rapamycin inhibitor, and were independent of the phosphatase and tensin homolog status. This highlights the potential relevance of metformin in combinatorial settings for the treatment of breast cancer.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

The morphology and composition of subnuclear organelles, such as Cajal bodies (CBs), nucleoli, and other nuclear bodies, is dynamic and can change in response to a variety of cell stimuli, including stress. We show that UV-C irradiation disrupts CBs and alters the distribution of a specific subset of CB components. The effect of UV-C on CBs differs from previously reported effects of transcription inhibitors. We demonstrate that the mechanism underlying the response of CBs to UV-C is mediated, at least in part, by PA28gamma (proteasome activator subunit gamma). The presence of PA28gamma in coilin-containing complexes is increased by UV-C. Overexpression of PA28gamma, in the absence of UV-C treatment, provokes a similar redistribution of the same subset of CB components that respond to UV-C. RNA interference-mediated knockdown of PA28gamma attenuates the nuclear disruption caused by UV-C. These data demonstrate that CBs are specific nuclear targets of cellular stress-response pathways and identify PA28gamma as a novel regulator of CB integrity.

This review surveys what is known about the structure and function of the subnuclear domains called Cajal bodies (CBs). The major focus is on CBs in mammalian cells but we provide an overview of homologous CB structures in other organisms. We discuss the protein and RNA components of CBs, including factors recently found to associate in a cell cycle-dependent fashion or under specific metabolic or stress conditions. We also consider the dynamic properties of both CBs and their molecular components, based largely on recent data obtained thanks to the advent of improved in vivo detection and imaging methods. We discuss how these data contribute to an understanding of CB functions and highlight major questions that remain to be answered. Finally, we consider the interesting links that have emerged between CBs and alterations in nuclear structure apparent in a range of human pathologies, including cancer and inherited neurodegenerative diseases. We speculate on the relationship between CB function and molecular disease.

A general strategy for inactivation of target proteins is presented, which we have termed “oligomerization chain reaction.” This technique is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally. Oligomerization through the CC region of promyelocytic leukemia, and through the natural self-associating domain, triggers the oligomerization chain reaction, leading to formation of large molecular weight complexes and functional inactivation of the target. As a test case, we have chosen the oncosuppressor p53, naturally occurring as a tetramer. Fusion of the CC to p53 leads to formation of stable high molecular weight complexes—as shown by size exclusion chromatography—to which wild-type p53 is recruited with high efficiency. CC-p53 chimeras delocalize wild-type p53 to the cytoplasm and inhibit its transcriptional regulatory properties, resulting in a loss of p53 function. We propose that this strategy may be of general application to self-associating factors and represent a complementary approach to currently used functional inactivation-based strategies.

Clinical management of malignant pleural mesothelioma (MPM) is very challenging because of the uncommon resistance of this tumor to chemotherapy. We report here increased expression of macrophage colony-stimulating-factor-1-receptor (M-CSF/CSF-1R) mRNA in mesothelioma versus normal tissue specimens and demonstrate that CSF-1R expression identifies chemoresistant cells of mesothelial nature in both primary cultures and mesothelioma cell lines. By using RNAi or ligand trapping, we demonstrate that the chemoresistance properties of those cells depend on autocrine CSF-1R signaling. At the single-cell level, the isolated CSF-1Rpos cells exhibit a complex repertoire of pluripotency, epithelial–mesenchymal transition and detoxifying factors, which define a clonogenic, chemoresistant, precursor-like cell sub-population. The simple activation of CSF-1R in untransformed mesothelial cells is sufficient to confer clonogenicity and resistance to pemetrexed, hallmarks of mesothelioma. In addition, this induced a gene expression profile highly mimicking that observed in the MPM cells endogenously expressing the receptor and the ligands, suggesting that CSF-1R expression is mainly responsible for the phenotype of the identified cell sub-populations. The survival of CSF1Rpos cells requires active AKT (v-akt murine thymoma viral oncogene homolog 1) signaling, which contributed to increased levels of nuclear, transcriptionally competent β-catenin. Inhibition of AKT reduced the transcriptional activity of β-catenin-dependent reporters and sensitized the cells to senescence-induced clonogenic death after pemetrexed treatment. This work expands what is known on the non-macrophage functions of CSF-1R and its role in solid tumors, and suggests that CSF-1R signaling may have a critical pathogenic role in a prototypical, inflammation-related cancer such as MPM and therefore may represent a promising target for therapeutic intervention.

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets. Background: IL-34 and the known IL-34 receptor, CSF-1R, are differentially expressed in mouse brain; thus, IL-34 may signal via an additional receptor(s). Results: IL-34 binds to PTP-ζ on U251 human glioblastoma cells to stimulate intracellular signaling and responses. Conclusion: PTP-ζ is an IL-34 receptor. Significance: CSF-1R-independent actions of IL-34 via PTP-ζ should be considered in evaluating IL-34 roles in development and disease.

Chronic inflammation appears to be a driving force behind cancer progression. NFκB and STAT3 activation plays a pertinent role in this process by mediating chemoresistance and the acquisition of mesenchymal features of protumorigenic cells. Epidemiological data and experimental observations suggest that Malignant Pleural Mesothelioma (MPM) is a prototype of chronic inflammation-driven cancer. Chemoresistance is a major feature of MPM. Thus, this paper explores the effect of butein (3,4,2',4'-tetrahydroxychalcone), a naturally occurring NFκB and STAT3 inhibitor, on the tumorigenic properties of MPM cells. MPM cells harbor high nuclear levels of NFκB and pSTAT3(Y(705)). Butein inhibits pSTAT3(Y(705)) phosphorylation, nuclear localization of NFκB and the physical interaction of NFκB and pSTAT3. This correlates with a downregulation of several genes involved in cancer progression (such as ICAM1, Vimentin, MMP9, Twist) of proangiogenic cytokines (VEGF) and of IL-6 and IL-8, key growth factors for MPM. Hence, butein inhibits the migration of MPM cells and strongly affects the clonogenicity of MPM cells in vitro. Finally, we show that the in vitro actions of butein translate into anticancer effects in vivo. In fact, butein treatment severely affects tumor engraftment and potentiates the anticancer effects of pemetrexed in mouse xenograft models. Butein does not significantly affect the viability of human, untransformed mesothelial cells in vitro, nor does it affect survival of tumor-free mice in vivo. The possibility of using butein as an additional treatment to current MPM therapies is discussed here.

Here we show that a subpopulation of MCF-7 breast cancer cells which stains pale to Toluidine Blue (Light Cells- LCs), is endowed with features of CSCs. LCs give rise to self-renewing mammospheres and express typical CSC markers; moreover this subpopulation is chemoresistant and highly tumorigenic in vivo. LCs can be identified in several other breast cancer cell lines, irrespectively of their histological origin (luminal vs. mesenchymal vs. basal) and represent an heterogeneous cell population composed mainly of CSC-like and early progenitor cells. By a limited in vitro drug screening assay, we identify compounds which can specifically interfere with the viability of LCs from multiple breast cancer cell lines. Analysis of the Sphere-Forming Efficiency (SFE) and of the distribution of ALDH(bright) cells within the treated cell lines suggest that one of the identified compounds acts in vitro by modulating the CSC phenotype. Interestingly, a subset of the identified compounds is known to affect directly or indirectly the NFkappaB pathway which is emerging as an important modulator of CSC proliferation and chemoresistance.

In this issue of Molecular Cell , Sen et al. (2011) report the involvement of PGC1α in modulating the transcriptional activity of p53 in metabolically challenged cells. They provide important insights into the mechanisms linking length and strength of the metabolic stress stimuli to the specific activation of p53 target genes.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

In the last years, two independent concepts have improved our understanding of cancer recurrence and spread: (1) the cancer stem cell (CSC) hypothesis and (2) the occurrence of epithelial-to-mesenchymal transition (EMT). Recent evidences, such as the one presented in a recent issue by Borgna et al.,1 point to a line of convergence of the two concepts. EMT is a physiological cell reprogramming event utilized in tissue remodeling during embryonic development and activated in normal adult tissues during regeneration.2 The presence of EMT-like cells in tumors has been linked to increased invasive and metastatic properties. The CSC hypothesis postulates the existence of hierarchically high-positioned, chemoresistant cells, which are responsible for disease relapse after treatment with debulking agents. These cells are endowed with the ability to reconstitute the histological heterogeneity of the originating tumor upon transplantation in immunodeficient hosts.3 These properties of CSCs have strict resemblance to tissue remodeling and repair, which are typical features of mesenchymal tissues. Indeed, it has been shown that cultured breast cells that have undergone EMT in vitro also possess cancer stem cell signatures and properties.4 Growth of breast cancer cells as non-adherent spheroids in relatively non-differentiating conditions is regarded as a useful tool to enrich cells endowed with CSC-like features, such as chemoresistance and tumor-repopulating ability.5 In the November 2012 issue of Cell Cycle, Silvia Borgna and colleagues provide evidence, at a molecular level and by using a large panel of cell lines corresponding to different breast cancer subtypes, that mammosphere-inducing growth conditions enrich for EMT-like cell subpopulations as well.1 This is especially true for Claudin-low breast cancer cell lines, which are highly enriched for CSC-like, CD44high/CD24low cells. Their work once more suggests that acquisition of EMT and CSC features are highly interconnected processes, possibly relevant for the organization of mammospheres. Indeed, recent evidence has been provided that the interaction of cell subpopulations with distinct mesenchymal and epithelial traits is instrumental for the maintenance of CSC-like cells6 and relies upon cytokine-mediated signaling7 (Fig. 1). In light of this, it may be worth noticing that mammospheres are heterogeneous in composition and represent an ideal place for paracrine signaling to occur between different cell subpopulations. One may thus predict that compounds interfering with this crosstalk can block mammosphere formation (Fig. 1). Indeed Butein, a naturally occurring STAT3 and NFκB inhibitor, impairs mammosphere formation from multiple breast cancer cell lines,5 possibly by blocking IL-6 signaling.6 Figure 1. Schematic hypothetical working model. Mammospheres contain both CSC and EMT-like cells. When grown as mammospheres in non-adherent and no-serum conditions, breast cancer cell cultures are progressively enriched in EMT-like cells. Inset: ... Interestingly, Borgna et al. found at least one of the known EMT-promoting transcription factors to be dynamically modulated in most of their cultures in time (mainly SNAI2 and TWIST1) when shifting from adherent to mammosphere culture conditions.1 This underscores the relevance of such a process and its activation by distinct, converging and interconnected pathways. Indeed, forced expression of individual EMT-inducing transcription factors in stabilized cell lines has led Weinberg and collaborators to postulate the existence of an EMT interactome of transcription factors which are capable of reciprocally influencing each other. It will be interesting to evaluate the levels of EMT-promoting factors upon chemotherapy treatment of spheroids in vitro and to establish whether the enrichment for EMT-like cells is relevant to chemoresistance of mammospheres. Culturing cancer cells as 3D spheroids may represent, therefore, a simplified albeit very useful tool for reproducing in vitro transient dynamic states of the tumor growth. It may also stimulate a shift in the way we envision hunting for novel therapeutic tools. Finally, this methodology is of general value, as it can be applied to cancers from other histotypes. For example, cells derived from biopsies or from malignant pleural effusions of patients with NSCLC give rise efficiently to propagating tumor spheroids in culture,8 which are, again, enriched in CSC markers.8,9 In conclusion, in vitro cultures of tumor spheroids from stabilized cell lines and from fresh tumor specimens may therefore be considered a useful in vitro model to screen for new agents capable of co-targeting both CSCs or EMT malignant features of cancer cells.