A new study shows that the expression of two classes of repetitive elements in the mouse genome is controlled through two complementary mechanisms: DNA methylation and p53-mediated transcription suppression.¹ When both lines of defense fail, expression of the repeats yields large quantities of double-stranded RNA, triggering interferon response that leads to caspase-dependent cell death. These notable findings highlight two fundamental trends: tight coupling of defense and cell death mechanisms that appears to be universal in cellular life and the exploitation of the expression of “junk” DNA as a signal triggering “altruistic” cell suicide.
p53; transposable elements; SINE repeats; DNA methylation; interferon response
Prostate cancer (PCa) remains one of the most prevalent malignancies affecting men in the western world. The etiology for PCa development and molecular mechanisms underlying castration-resistant progression are incompletely understood. Emerging evidence from many tumor systems has shown the existence of distinct subpopulations of stem like-cancer cells termed cancer stem cells (CSCs), which may be involved in tumor initiation, progression, metastasis and therapy resistance. Prostate cancer stem cells (PCSCs) have also been identified using different experimental strategies in distinct model systems. In this brief review, we summarize our current knowledge of normal prostate stem/progenitor cells, highlight recent progress on PCSCs, expound on the potential cell-of-origin for PCa and discuss the involvement of PCSCs in PCa progression and castration resistance. Elucidation of the phenotypic and functional properties and molecular regulation of PCSCs will help us better understand PCa biology and may lead to development of novel therapeutics targeting castration-resistant PCa cells.
prostate cancer; cancer stem cells; prostate cancer stem cells; differentiation; therapy resistance
The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.
Ku80; Ku86; Ku70; SCF; DNA damage; double-strand break; nonhomologous end joining; Fbxl12; Fbl12; ubiquitin
Cyclin E1 is expressed at the G₁/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G₂M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.
cyclin E2; cell cycle; cyclin E1; proliferation; Fbw7
Cyclins E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.
cyclin E2; genomic instability; mitosis; p107; cyclin E1
Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit γ-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1−/− cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.
pericentrin; centrosome; DNA damage response; mitosis; checkpoint
Non-small cell lung carcinoma (NSCLC) is the most common form of lung cancer and is associated with a high mortality rate worldwide. The majority of individuals bearing NSCLC are treated with surgery plus adjuvant cisplatin, an initially effective therapeutic regimen that, however, is unable to prevent relapse within 5 years after tumor resection in an elevated proportion of patients. The factors that predict the clinical course of NSCLC and its sensitivity to therapy remain largely obscure. One notable exception is provided by pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. PDXK has recently been shown to be required for optimal cisplatin responses in vitro and in vivo and to constitute a bona fide prognostic marker in the NSCLC setting. Together with PDXK, 84 additional factors were identified that influence the response of NSCLC cells to cisplatin, in vitro including the hepatic lipase LIPC. Here, we report that the intratumoral levels of LIPC, as assessed by immunohistochemistry in two independent cohorts of NSCLC patients, positively correlate with disease outcome. In one out of two cohorts studied, the overall survival of NSCLC patients bearing LIPChigh lesions was unaffected, if not slightly worsened, by cisplatin-based adjuvant therapy. Conversely, the overall survival of patients with LIPClow lesions was prolonged by post-operative cisplatin. Pending validation in appropriate clinical series, these results suggest that LIPClow NSCLC patients would be those who mainly benefit from adjuvant cisplatin therapy. Thus, the expression levels of LIPC appear to have an independent prognostic value (and perhaps a predictive potential) in the setting of NSCLC. If these findings were confirmed by additional studies, LIPC expression levels might allow not only for NSCLC patient stratification, but also for the implementation of personalized therapeutic approaches.
anaplastic lymphoma kinase; apoptosis; BCL-XL; PDXP; personalized medicine; pyridoxine
Primary cilia are microtubule-based solitary sensing structures on the cell surface that play crucial roles in cell signaling and development. Abnormal ciliary function leads to various human genetic disorders, collectively known as ciliopathies. Outer dense fiber protein 2 (Odf2) was initially isolated as a major component of sperm-tail fibers. Subsequent studies have demonstrated the existence of many splicing variants of Odf2, including Cenexin1 (Odf2 isoform 9), which bears an unusual C-terminal extension. Strikingly, Odf2 localizes along the axoneme of primary cilia, whereas Cenexin1 localizes to basal bodies in cultured mammalian cells. Whether Odf2 and Cenexin1 contribute to primary cilia assembly by carrying out either concerted or distinct functions is unknown. By taking advantage of odf2−/− cells lacking endogenous Odf2 and Cenexin1, but exogenously expressing one or both of these proteins, we showed that Cenexin1, but not Odf2, was necessary and sufficient to induce ciliogenesis. Furthermore, the Cenexin1-dependent primary cilia assembly pathway appeared to function independently of Odf2. Consistently, Cenexin1, but not Odf2, interacted with GTP-loaded Rab8a, localized to the distal/subdistal appendages of basal bodies, and facilitated the recruitment of Chibby, a centriolar component that is important for proper ciliogenesis. Taken together, our results suggest that Cenexin1 plays a critical role in ciliogenesis through its C-terminal extension that confers a unique ability to mediate primary cilia assembly. The presence of multiple splicing variants hints that the function of Odf2 is diversified in such a way that each variant has a distinct role in the complex cellular and developmental processes.
Cenexin1; Odf2; Rab8a; Chibby; primary cilia; ciliogenesis
Aurora B kinase is an integral regulator of cytokinesis as it stabilizes the intercellular canal within the midbody to ensure proper chromosomal segregation during cell division. Here we identified an E3 ligase subunit, F box protein FBXL2, that by recognizing a calmodulin binding signature within Aurora B, ubiquitinates and removes the kinase from the midbody. Calmodulin, by competing with the F box protein for access to the calmodulin binding signature, protected Aurora B from FBXL2. Calmodulin co-localized with Aurora B on the midbody, preserved Aurora B levels in cells, and stabilized intercellular canals during delayed abscission. Genetic or pharmaceutical depletion of endogenous calmodulin significantly reduced Aurora B protein levels at the midbody resulting in tetraploidy and multi-spindle formation. The calmodulin inhibitor, calmidazolium, reduced Aurora B protein levels resulting in tetraploidy, mitotic arrest, and apoptosis of tumorigenic cells and profoundly inhibiting tumor formation in athymic nude mice. These observations indicate molecular interplay between Aurora B and calmodulin in telophase and suggest that calmodulin acts as a checkpoint sensor for chromosomal segregation errors during mitosis.
Aurora B; FBXL2; calmodulin; mitosis; midbody
The term “mitochondrial permeability transition” (MPT) refers to an abrupt increase in the permeability of the inner mitochondrial membrane to low molecular weight solutes. Due to osmotic forces, MPT is paralleled by a massive influx of water into the mitochondrial matrix, eventually leading to the structural collapse of the organelle. Thus, MPT can initiate mitochondrial outer membrane permeabilization (MOMP), promoting the activation of the apoptotic caspase cascade as well as of caspase-independent cell death mechanisms. MPT appears to be mediated by the opening of the so-called “permeability transition pore complex” (PTPC), a poorly characterized and versatile supramolecular entity assembled at the junctions between the inner and outer mitochondrial membranes. In spite of considerable experimental efforts, the precise molecular composition of the PTPC remains obscure and only one of its constituents, cyclophilin D (CYPD), has been ascribed with a crucial role in the regulation of cell death. Conversely, the results of genetic experiments indicate that other major components of the PTPC, such as voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT), are dispensable for MPT-driven MOMP. Here, we demonstrate that the c subunit of the FO ATP synthase is required for MPT, mitochondrial fragmentation and cell death as induced by cytosolic calcium overload and oxidative stress in both glycolytic and respiratory cell models. Our results strongly suggest that, similar to CYPD, the c subunit of the FO ATP synthase constitutes a critical component of the PTPC.
ATP5G1; apoptosis; caspases; cytochrome c; mitochondrial respiratory chain; p53; permeability transition pore (PTP)
Our previous reports showed that the cisplatin exposure induced the ATM-dependent phosphorylation of ΔNp63a, which is subsequently involved in transcriptional regulation of gene promoters encoding mRNAs and microRNAs in squamous cell carcinoma (SCC) cells upon cisplatin-induced cell death. We showed that phosphorylated (p)-ΔNp63a plays a role in upregulation of pro-apoptotic proteins, while non-p-ΔNp63a is implicated in pro-survival signaling. In contrast to non-p-ΔNp63a, p-ΔNp63a modulated expression of specific microRNAs in SCC cells exposed to cisplatin. These microRNAs were shown to attenuate the expression of several proteins involved in cell death/survival, suggesting the critical role for p-ΔNp63a in regulation of tumor cell resistance to cisplatin. Here, we studied the function of ΔNp63a in transcriptional activation and repression of the specific microRNA promoters whose expression is affected by cisplatin treatment of SCC cells. We quantitatively studied chromatin-associated proteins bound to tumor protein (TP) p63-responsive element, we found that p-ΔNp63a along with certain transcription coactivators (e.g., CARM1, KAT2B, TFAP2A, etc.) necessary to induce gene promoters for microRNAs (630 and 885-3p) or with transcription corepressors (e.g., EZH2, CTBP1, HDACs, etc.) needed to repress promoters for microRNAs (181a-5p, 374a-5p and 519a-3p) in SCC cells exposed to cisplatin.
p53; p63; cisplatin; squamous cell carcinomas; DNA/protein interactions; microRNAs
The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.
p53; mdm2; nutlin; Brownian dynamics; residence time; encounter complex; basins of attraction
Circadian rhythms control multiple physiological and pathological processes, including embryonic development in mammals and development of various human diseases. We have recently, in a developing zebrafish embryonic model, discovered that the circadian oscillation controls developmental angiogenesis. Disruption of crucial circadian regulatory genes, including Bmal1 and Period2, results in marked impairment or enhancement of vascular development in zebrafish. At the molecular level, we show that the circadian regulator Bmal1 directly targets the promoter region of the vegf gene in zebrafish, leading to an elevated expression of VEGF. These findings can reasonably be extended to developmental angiogenesis in mammals and even pathological angiogenesis in humans. Thus, our findings, for the first time, shed new light on mechanisms that underlie circadian clock-regulated angiogenesis.
zebrafish; circadian; angiogenesis; Bmal1; Period2; VEGF; Clock; development; vasculogenesis; vasculature
Vitamin B6 metabolism influences the adaptive response of non-small lung carcinoma (NSCLC) cells to distinct, potentially lethal perturbations in homeostasis, encompassing nutrient deprivation, hyperthermia, hypoxia, irradiation as well as the exposure to cytotoxic chemicals, including the DNA-damaging agent cisplatin (CDDP). Thus, the siRNA-mediated downregulation of pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6, protects NSCLC cells (as well as a large collection of human and murine malignant cells of distinct histological derivation) from the cytotoxic effects of CDDP. Accordingly, the administration of pyridoxine, one of the inactive precursors of vitamin B6, exacerbates cisplatin-induced cell death, in vitro and in vivo, but only when PDXK is expressed. Conversely, antioxidants such as non-oxidized glutathione (GSH) are known to protect cancer cells from CDDP toxicity. Pyridoxine increases the amount of CDDP-DNA adducts formed upon the exposure of NSCLC cells to CDDP and aggravates the consequent DNA damage response. On the contrary, in the presence of GSH, NSCLC cells exhibit near-to-undetectable levels of CDDP-DNA adducts and a small fraction of the cell population activates the DNA damage response. We therefore wondered whether vitamin B6 metabolism and GSH might interact with CDDP in a pharmacokinetic fashion. In this short communication, we demonstrate that GSH inhibits the intracellular accumulation of CDDP, while pyridoxine potentiates it in a PDXK-dependent fashion. Importantly, such pharmacokinetic effects do not involve plasma membrane transporters that mediate a prominent fraction of CDDP influx, i.e., solute carrier family 31, member 1 (SLC31A1, best known as copper transporter 1, CTR1) and efflux, i.e., ATPase, Cu2+ transporting, β polypeptide (ATP7B).
A549; apoptosis; N-acetyl-cysteine; PDXP; reactive oxygen species; Wilson disease
Heterochromatin protein 1 paralogs (HP1α, β and γ in mammals) are not only central in heterochromatin organization, but have also been linked to transcriptional activation at euchromatic regions, maintenance of telomere stability and, most recently, to the DNA damage response (DDR). However, how HP1 proteins contribute to the DDR at a molecular level, and whether HP1 paralogs within the same organism, as well as their respective orthologs, have overlapping or unique roles in the DDR, remain to be elucidated. Herein, we have combined the analysis of the efficiency and kinetics of recruitment of key repair proteins to sites of DNA damage with specific DNA repair assays to demonstrate that human HP1 paralogs differentially modulate homology-directed repair (HDR) pathways, including homologous recombination (HR) and single-strand annealing (SSA). We find that while HP1α and β stimulate HR and SSA, HP1γ has an inhibitory role. In addition, we show that the stimulatory role of HP1α and β in HDR is linked to the DNA-end resection step of DNA breaks, through the promotion of RPA loading and phosphorylation at damage sites. Altogether, our findings provide mechanistic insight into how human HP1 proteins participate in the recombination process, emerging as important chromatin regulators during HDR.
DNA damage response; DNA end resection; DNA repair; HP1; chromatin; epigenetics; homologous recombination; non-histone chromatin proteins
Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.
mesenchymal stem/stromal cells; transdifferentiation; cyclin-dependent kinase 4; neural cells; glial cells; cell cycle arrest; neurodegenerative disease
The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.
HIV; latency; Tat; JQ1; MS417; I-BET; I-BET151; P-TEFb; BRD4; BRD2
Circumstantial evidence suggests that colon carcinogenesis can ensue the transient tetraploidization of (pre-)malignant cells. In line with this notion, the tumor suppressors APC and TP53, both of which are frequently inactivated in colon cancer, inhibit tetraploidization in vitro and in vivo. Here, we show that—contrarily to their wild-type counterparts—Tp53−/− colonocytes are susceptible to drug-induced or spontaneous tetraploidization in vitro. Colon organoids generated from tetraploid Tp53−/− cells exhibit a close-to-normal morphology as compared to their diploid Tp53−/− counterparts, yet the colonocytes constituting these organoids are characterized by an increased cell size and an elevated expression of the immunostimulatory protein calreticulin on the cell surface. The subcutaneous injection of tetraploid Tp53−/− colon organoids led to the generation of proliferating tumors in immunodeficient, but not immunocompetent, mice. Thus, tetraploid Tp53−/− colonocytes fail to survive in immunocompetent mice and develop neoplastic lesions in immunocompromised settings only. These results suggest that tetraploidy is particularly oncogenic in the context of deficient immunosurveillance.
apoptosis; cell cycle; cytochalasin D; mitotic catastrophe; nocodazole; p53
Here, we provide the necessary proof of concept, that it is possible to metabolically create a non-permissive or “hostile” stromal microenvironment, which actively prevents tumor engraftment in vivo. We developed a novel genetically engineered fibroblast cell line that completely prevents tumor formation in mice, with a 100% protection rate. No host side effects were apparent. This could represent a viable cellular strategy for preventing and treating a variety of human cancers. More specifically, we examined the autocrine and paracrine effects of the cellular delivery of TNFα on breast cancer tumor growth and cancer metabolism. For this purpose, we recombinantly overexpressed TNFα in human breast cancer cells (MDA-MB-231) or human immortalized fibroblasts (hTERT-BJ1). Our results directly show that TNFα functions as a potent tumor suppressor. Remarkably, TNFα-expressing breast cancer cells were viable, without any significant increases in their basal apoptotic rate. However, after 4 weeks post-implantation, TNFα-expressing breast cancer cells failed to form any tumors in xenografted mice (0 tumors/10 injections), ultimately conferring 100% protection against tumorigenesis. Similarly, TNFα-overexpressing fibroblasts were also viable, without any increases in apoptosis. Significantly, complete tumor suppression was obtained by co-injecting TNFα expressing stromal fibroblasts with human breast cancer cells, indicating that paracrine cell-mediated delivery of TNFα can also prevent tumor engraftment and growth (0 tumors/10 injections). Mechanistically, TNFα induced autophagy and mitochondrial dysfunction in both epithelial cancer cells and stromal fibroblasts, preventing energy transfer from the tumor microenvironment, likely “starving” the cancer cells to death. In addition, via qRT-PCR analysis of MDA-MB-231 cells, we observed that TNFα mediated the upregulation of gene transcripts associated with inflammation and senescence [IL-1-β, IL-6, IL-8, MCP-1, COX-2, p21(WAF1/CIP1)] and downregulated known tumor-promoting genes (collagen VI and MMP2). Recombinant overexpression of TNFα receptor(s) in MDA-MB-231 cells also significantly reduced tumor growth, but was not as effective as the TNFα ligand itself in preventing tumor growth. Thus, we propose that stromal cell-mediated delivery of TNFα to human tumors [using transfected fibroblasts or mesenchymal stem cells (hMSCs)] may be a novel and effective strategy for the prevention and treatment of human cancers.
tumor necrosis factor (TNF); cancer prevention; cellular therapy; fibroblast mediated delivery; mitochondrial dysfunction; breast cancer; tumor growth; tumor cell engraftment; autophagy; apoptosis
Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.
glioblastoma; sorafenib; therapy; tumor initiating cells; stemness; Mcl-1
Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a key role in the development of atherosclerosis. However, the mechanisms that mediate VSMC phenotypic switching are unclear. We report here that TIPE2, the tumor necrosis factor (TNF) α-induced protein 8-like 2 (TNFAIP8L2), plays an atheroprotective role by regulating phenotypic switching of VSMCs in response to oxidized low-density lipoprotein (ox-LDL) stimuli. TIPE2-deficient VSMCs treated with ox-LDL expressed lower levels of contractile proteins such as SMαA, SM-MHC and calponin, whereas the proliferation, migration and the synthetic capacity for growth factors and cytokines were increased remarkably. Furthermore, TIPE2 inhibited VSMCs proliferation by preventing G1/S phase transition. Interestingly, these effects of TIPE2 on VSMCs were dependent on P38 and ERK1/2 kinase signals. As a result, neointima formation was accelerated in the carotid arteries of TIPE2-deficient mice. These results indicate that TIPE2 is a potential inhibitor of atherosclerosis.
TNFAIP8L2 (TIPE2); vascular smooth muscle cell; phenotypic switching; cell cycle; atherosclerosis
Primary ovarian cancer is responsive to treatment, but chemoresistant recurrent disease ensues in majority of patients. Recent compelling evidence demonstrates that a specific population of cancer cells, the cancer stem cells, initiates and sustains tumors. It is therefore possible that this cell population is also responsible for recurrence. We have shown previously that CD44+/MyD88+ epithelial ovarian cancer stem cells (CD44+/MyD88+ EOC stem cells) are responsible for tumor initiation. In this study, we demonstrate that this population drives tumor repair following surgery- and chemotherapy-induced tumor injury. Using in vivo and in vitro models, we also demonstrate that during the process of tumor repair, CD44+/MyD88+ EOC stem cells undergo self-renewal as evidenced by upregulation of stemness-associated genes. More importantly, we show that a pro-inflammatory microenvironment created by the TLR2-MyD88-NFκB pathway supports EOC stem cell-driven repair and self-renewal. Overall, our findings point to a specific cancer cell population, the CD44+/MyD88+ EOC stem cells and a specific pro-inflammatory pathway, the TLR2-MyD88-NFκB pathway, as two of the required players promoting tumor repair, which is associated with enhanced cancer stem cell load. Identification of these key players is the first step in elucidating the steps necessary to prevent recurrence in EOC patients.
ovarian cancer stem cells; recurrence; tumor repair; TLR2; self-renewal
Mutant K-Ras and survivin both contribute to oncogenesis, but little is known about K-Ras requirement for the maintenance of the high levels of survivin in human tumors. Here we demonstrate that K-Ras depletion significantly decreases survivin levels in human cancer cells that harbor mutant but not wild type K-Ras. K-Ras depletion attenuates both basal and drug-induced survivin levels. The mechanism by which K-Ras depletion decreases survivin levels is through ubiquitination and proteasomal degradation of survivin and is independent of survivin-Thr-34 phosphorylation. Depletion of RalA and RalB, but not Raf-1, Akt1 and Akt2, decreases survivin levels, suggesting that K-Ras may regulate survivin stability through its RalGDS/Ral but not PI3K/Akt and Raf-1/Mek effector pathways. Furthermore, the ability of mutant K-Ras to induce anchorage-independent growth, invasion and survival is compromised by depletion of survivin. These studies suggest that mutant K-Ras contributes to the maintenance of the aberrantly high levels of survivin in tumors by regulating its stability, and that the ability of mutant K-Ras to induce malignant transformation is, at least in part, dependent on these high levels of survivin.
K-Ras; Survivin; apoptosis; cancer; proteasome; protein degradation
Induced pluripotent stem (iPS) cells share some basic properties, such as self-renewal and pluripotency, with cancer cells, and they also appear to share several metabolic alterations that are commonly observed in human tumors. The cancer cells’ glycolytic phenotype, first reported by Otto Warburg, is necessary for the optimal routing of somatic cells to pluripotency. However, how iPS cells establish a Warburg-like metabolic phenotype and whether the metabolic pathways that support the bioenergetics of iPS cells are produced by the same mechanisms that are selected during the tumorigenic process remain largely unexplored. We recently investigated whether the reprogramming-competent metabotype of iPS cells involves changes in the activation/expression status of the H+-ATPase, which is a core component of mitochondrial oxidative phosphorylation that is repressed at both the activity and protein levels in human carcinomas, and of the lipogenic switch, which refers to a marked overexpression and hyperactivity of the acetyl-CoA carboxylase (ACACA) and fatty acid synthase (FASN) lipogenic enzymes that has been observed in nearly all examined cancer types. A comparison of a starting population of mouse embryonic fibroblasts and their iPS cell progeny revealed that somatic cell reprogramming involves a significant increase in the expression of ATPase inhibitor factor 1 (IF1), accompanied by extremely low expression levels of the catalytic β-F1-ATPase subunit. The pharmacological inhibition of ACACA and FASN activities markedly decreases reprogramming efficiency, and ACACA and FASN expression are notably upregulated in iPS cells. Importantly, iPS cells exhibited a significant intracellular accumulation of neutral lipid bodies; however, these bodies may be a reflection of intense lysosomal/autophagocytic activity rather than bona fide lipid droplet formation in iPS cells, as they were largely unresponsive to pharmacological modulation of PPARgamma and FASN activities. The AMPK agonist metformin, which endows somatic cells with a bioenergetic infrastructure that is protected against reprogramming, was found to drastically elongate fibroblast mitochondria, fully reverse the high IF1/β-F1-ATPase ratio and downregulate the ACACA/FASN lipogenic enzymes in iPS cells. The mitochondrial H+-ATP synthase and the ACACA/FASN-driven lipogenic switch are newly characterized as instrumental metabolic events that, by coupling the Warburg effect to anabolic metabolism, enable de-differentiation during the reprogramming of somatic cells to iPS cells.
iPS cells; bioenergetics; Warburg effect; mitochondria; glycolysis; H+-ATP synthase; IF1; fatty acid synthase; acetyl CoA carboxylase; lipogenesis; SSEA1