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Tubulin-binding agents such as taxol, vincristine or vinblastine are well-established drugs in clinical treatment of metastatic cancer. However, because of their highly complex chemical structures, the synthesis and hence the supply issues are still quite challenging. Here we set on stage pretubulysin, a chemically accessible precursor of tubulysin that was identified as a potent microtubule-binding agent produced by myxobacteria. Although much simpler in chemical structure, pretubulysin abrogates proliferation and long-term survival as well as anchorage-independent growth, and also induces anoikis and apoptosis in invasive tumor cells equally potent to tubulysin. Moreover, pretubulysin posseses in vivo efficacy shown in a chicken chorioallantoic membrane (CAM) model with T24 bladder tumor cells, in a mouse xenograft model using MDA-MB-231 mammary cancer cells and finally in a model of lung metastasis induced by 4T1 mouse breast cancer cells. Pretubulysin induces cell death via the intrinsic apoptosis pathway by abrogating the expression of pivotal antiapoptotic proteins, namely Mcl-1 and Bcl-xL, and shows distinct chemosensitizing properties in combination with TRAIL in two- and three-dimensional cell culture models. Unraveling the underlying signaling pathways provides novel information: pretubulysin induces proteasomal degradation of Mcl-1 by activation of mitogen-activated protein kinase (especially JNK (c-Jun N-terminal kinase)) and phosphorylation of Mcl-1, which is then targeted by the SCF(Fbw7) E3 ubiquitin ligase complex for ubiquitination and degradation. In sum, we designate the microtubule-destabilizing compound pretubulysin as a highly promising novel agent for mono treatment and combinatory treatment of invasive cancer.
We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.
The ability to escape apoptosis or programmed cell death is a hallmark of human cancers, for example pancreatic cancer. This can promote tumorigenesis, since too little cell death by apoptosis disturbs tissue homeostasis. Additionally, defective apoptosis signaling is the underlying cause of failure to respond to current treatment approaches, since therapy-mediated antitumor activity requires the intactness of apoptosis signaling pathways in cancer cells. Thus, the elucidation of defects in the regulation of apoptosis in pancreatic carcinoma can result in the identification of novel targets for therapeutic interference and for exploitation for cancer drug discovery. Keywords: apoptosis; pancreatic cancer; TRAIL; IAPs; mitochondria
Novel insights into the synergistic interaction of Bortezomib and TRAIL: tBid provides the link
(2011)
The proteasome inhibitor Bortezomib has been identified as a potent enhancer of TRAIL-induced apoptosis in several human cancers. However, the identification of the underlying molecular mechanisms of this synergistic cell death induction has been ongoing over the last years. A recent study identifies a new mechanism of action for the synergism of TRAIL and Bortezomib.
Rhabdomyosarcomas are the most common pediatric soft tissue sarcoma and are a major cause of death from cancer in young patients requiring new treatment options to improve outcomes. High-risk patients include those with metastatic or relapsed disease and tumors with PAX3-FOXO1 fusion genes that encode a potent transcription factor that drives tumourigenesis through transcriptional reprogramming. Polo-Like Kinase-1 (PLK1) is a serine/threonine kinase that phosphorylates a wide range of target substrates and alters their activity. PLK1 functions as a pleiotropic master regulator of mitosis and regulates DNA replication after stress. Taken together with high levels of expression that correlate with poor outcomes in many cancers, including rhabdomyosarcomas, it is an attractive therapeutic target. This is supported in rhabdomyosarcoma models by characterization of molecular and phenotypic effects of reducing and inhibiting PLK1, including changes to the PAX3-FOXO1 fusion protein. However, as tumor re-growth has been observed, combination strategies are required. Here we review preclinical evidence and consider biological rationale for PLK1 inhibition in combination with drugs that promote apoptosis, interfere with activity of PAX3-FOXO1 and are synergistic with microtubule-destabilizing drugs such as vincristine. The preclinical effects of low doses of the PLK1 inhibitor volasertib in combination with vincristine, which is widely used in rhabdomyosarcoma treatment, show particular promise in light of recent clinical data in the pediatric setting that support achievable volasertib doses predicted to be effective. Further development of novel therapeutic strategies including PLK1 inhibition may ultimately benefit young patients with rhabdomyosarcoma and other cancers.
Secondary plant metabolites reveal numerous biological activities making them attractive as resource for drug development of human diseases. As the majority of cancer drugs clinically established during the past half century is derived from nature, cancer researchers worldwide try to identify novel natural products as lead compounds for cancer therapy. Natural products are considered as promising cancer therapeutics, either as single agents or in combination protocols, to enhance the antitumor activity of additional therapeutic modalities. Most natural compounds exert pleotrophic effects and modulate various signal transduction pathways. A better understanding of the complex mechanisms of action of natural products is expected to open new perspectives in coming years for their use alone or in combination therapies in oncology. Two major strategies to identify novel drug candidates from nature are the bioactivity-guided fractionation of medicinal plant extracts to isolate cytotoxic chemicals and the identification of small molecules inhibiting specific targets in cancer cells. In the present review, we report on our own efforts to unravel the molecular modes of action of phytochemicals in cancer cells and focus on resveratrol, betulinic acid, artesunate, dicentrine and camptothecin derivatives.
Autophagy in cancer therapy
(2017)
Autophagy represents a catabolic program involved in the degradation of cellular components via lysosomes. It serves to mitigate cellular stress and to provide metabolic precursors especially upon starvation. Thereby, autophagy can support the survival of cancer cells. In addition, there is now convincing evidence showing that under certain conditions autophagy can also foster cell death. This dual function of autophagy is also relevant upon anticancer treatment, as many chemotherapeutic agents engage autophagy. A better understanding of the molecular mechanisms that are critical for mediating autophagic cell death in cancer cells will be instrumental to selectively interfere with this cellular program in order to increase the cancer cell’s response to cytotoxic drugs. This review illustrates how anticancer drug-induced autophagy is involved in mediating cell death.
Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.
Different response of Ptch mutant and Ptch wildtype Rhabdomyosarcoma toward SMO and PI3K inhibitors
(2018)
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.
Smac (second mitochondria-derived activator of caspase) mimetics are considered as promising anticancer therapeutics and used to induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are often abundantly expressed in cancer cells. Here, we identify interferon regulatory factor 1 (IRF1) as a novel critical regulator of Smac mimetic BV6-induced apoptosis and proinflammatory cytokine secretion with impact on the immune response. IRF1 knockdown rescues cells from BV6-induced apoptosis and attenuates BV6-stimulated upregulation of tumor necrosis factor-α (TNFα), indicating that IRF1 mediates BV6-triggered cell death, at least in part, by inducing TNFα. This notion is supported by data showing that exogenous supply of TNFα restores BV6-induced cell death in IRF-knockdown cells. Interestingly, IRF1 selectively controls the induction of nuclear factor-κB (NF-κB) target genes, as IRF1 depletion attenuates BV6-stimulated upregulation of TNFα and interleukin-8 (IL-8) but not p100 and RelB. Concomitant knockdown of IRF1 and p65 cooperate to inhibit BV6-induced cell death, implying a cooperative interaction of IRF1 and NF-κB. In addition, IRF1 silencing hampers TNFα induction by TNFα itself as an another prototypical NF-κB stimulus. Importantly, IRF1 depletion impedes BV6-stimulated secretion of additional proinflammatory cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IL-6 and monocyte chemoattractant protein-1, and migration of primary monocytes to BV6-treated tumor cells. In conclusion, this identification of IRF1 as a dual regulator of BV6-induced apoptosis and inflammatory cytokine secretion provides novel insights into determinants of sensitivity towards Smac mimetic and possible implications of Smac mimetic treatment on tumor microenvironment and immune response.
Since Inhibitor of Apoptosis (IAP) proteins have been implicated in cellular adaptation to endoplasmic reticulum (ER) stress, we investigated the regulation of ER stress-induced apoptosis by small-molecule second mitochondria-derived activator of caspase (Smac) mimetics that antagonize IAP proteins. Here, we discover that Smac mimetic suppresses tunicamycin (TM)-induced apoptosis via resolution of the unfolded protein response (UPR) and ER stress. Smac mimetics such as BV6 selectively inhibit apoptosis triggered by pharmacological or genetic inhibition of protein N-glycosylation using TM or knockdown of DPAGT1, the enzyme that catalyzes the first step of protein N-glycosylation. In contrast, BV6 does not rescue cell death induced by other typical ER stressors (i.e., thapsigargin (TG), dithiothreitol, brefeldin A, bortezomib, or 2-deoxyglucose). The protection from TM-triggered apoptosis is found for structurally different Smac mimetics and for genetic knockdown of cellular IAP (cIAP) proteins in several cancer types, underlining the broader relevance. Interestingly, lectin microarray profiling reveals that BV6 counteracts TM-imposed inhibition of protein glycosylation. BV6 consistently abolishes TM-stimulated accumulation of ER stress markers such as glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and reduces protein kinase RNA-like ER kinase (PERK) phosphorylation and X box-binding protein 1 (XBP1) splicing upon TM treatment. BV6-stimulated activation of nuclear factor-κB (NF-κB) contributes to the resolution of ER stress, since NF-κB inhibition by overexpression of dominant-negative IκBα superrepressor counteracts the suppression of TM-stimulated transcriptional activation of CHOP and GRP78 by BV6. Thus, our study is the first to show that Smac mimetic protects from TM-triggered apoptosis by resolving the UPR and ER stress. This provides new insights into the regulation of cellular stress responses by Smac mimetics.
Ferroptosis, a newly discovered form of cell death mediated by reactive oxygen species (ROS) and lipid peroxidation, has recently been shown to have an impact on various cancer types; however, so far there are only few studies about its role in hepatocellular carcinoma (HCC). The delicate equilibrium of ROS in cancer cells has found to be crucial for cell survival, thus increased levels may trigger ferroptosis in HCC.In our study, we investigated the effect of different ROS modulators and ferroptosis inducers on a human HCC cell line and a human hepatoblastoma cell line. We identified a novel synergistic cell death induction by the combination of Auranofin and buthionine sulfoxime (BSO) or by Erastin and BSO at subtoxic concentrations. We found a caspase-independent, redox-regulated cell death, which could be rescued by different inhibitors of ferroptosis. Both cotreatments stimulated lipid peroxidation. All these findings indicated ferroptotic cell death. Both cotreatments affected the canonical ferroptosis pathway through GPX4 downregulation. We also found an accumulation of Nrf2 and HO-1, indicating an additional effect on the non-canonical pathway. Our results implicate that targeting these two main ferroptotic pathways simultaneously can overcome chemotherapy resistance in HCC.
Background: In the present study, we aimed to investigate the effect of counteracting inhibitor of apoptosis (IAP) proteins using the small molecule Second Mitochondria-derived Activator of Caspase (SMAC) mimetic BV6 in combination with ionizing radiation on apoptosis, cell cycle regulation, DNA double-strand break (DSB) repair, three-dimensional (3D) clonogenic survival and expression of IAPs in colorectal carcinoma cells.
Material and methods: Colorectal cancer cell lines (HCT-15, HT-29, SW480) were subjected to BV6 treatment (0–4 μM) with or without irradiation (2–8 Gy, single dose) followed by MTT, Caspase 3/7 activity, γH2AX/53BP1 foci assays, AnnexinV staining, cell cycle analysis, 3D colony forming assays and Western blotting (cellular IAP1 (cIAP1) and cIAP2, Survivin, X-linked IAP (XIAP)).
Results: BV6 treatment decreased cell viability and significantly increased irradiation-induced apoptosis as analyzed by Caspase 3/7 activity, AnnexinV-positive and subG1 phase cells. While basal 3D clonogenic survival was decreased in a cell line-dependent manner, BV6 significantly enhanced cellular radiosensitivity of all cell lines in a concentration-dependent manner and increased the number of radiation-induced γH2AX/53BP1-positive foci. Western blot analysis revealed a markedly reduced cIAP1 expression at 4 h after BV6 treatment in all cell lines, a substantial reduction of XIAP expression in SW480 and HT-29 cells at 24 h and a slightly decreased cIAP2 expression in HCT-15 cells at 48 h after treatment. Moreover, single or double knockdown of cIAP1 and XIAP resulted in significantly increased residual γH2AX/53BP1-positive foci 24 h after 2 Gy and radiosensitization relative to control small interfering RNA (siRNA)-treated cells.
Conclusion: The SMAC mimetic BV6 induced apoptosis and hampered DNA damage repair to radiosensitize 3D grown colorectal cancer cells. Our results demonstrate IAP targeting as a promising strategy to counteract radiation resistance of colorectal cancer cells.
Second mitochondria-derived activator of caspase (Smac) mimetics are considered as promising anticancer therapeutics that are currently under investigation in early clinical trials. They induce apoptosis by antagonizing inhibitor of apoptosis proteins, which are frequently overexpressed in cancer. We previously reported that Smac mimetics, such as BV6, additionally exert non-apoptotic functions in glioblastoma (GBM) cells by stimulating migration and invasion in a nuclear factor kappa B (NF-κB)-dependent manner. Because NF-κB target genes mediating these effects are largely unknown, we performed whole-genome expression analyses. Here, we identify chemokine (C-C motif) ligand 2 (CCL2) as the top-listed NF-κB-regulated gene being upregulated upon BV6 treatment in GBM cells. BV6-induced upregulation and secretion of CCL2 are required for migration and invasion of GBM cells because knockdown of CCL2 in GBM cells abolishes these effects. Co-culture experiments of GBM cells with non-malignant astroglial cells reveal that BV6-stimulated secretion of CCL2 by GBM cells into the supernatant triggers migration of astroglial cells toward GBM cells because CCL2 knockdown in BV6-treated GBM cells impedes BV6-stimulated migration of astroglial cells. In conclusion, we identify CCL2 as a BV6-induced NF-κB target gene that triggers migration and invasion of GBM cells and exerts paracrine effects on the GBM's microenvironment by stimulating migration of astroglial cells. These findings provide novel insights into the biological functions of Smac mimetics with important implications for the development of Smac mimetics as cancer therapeutics.
Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.
Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia–reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.
Rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) are among the most common pediatric sarcomas (Arndt et al., 2012). Despite sarcomas representing a highly heterogeneous group of tumors, ES and alveolar RMS (ARMS) typically share one common genetic characteristic, namely a specific chromosomal translocation (Helman and Meltzer, 2003; Lessnick and Ladanyi, 2012). These translocations generate fusion proteins, which are composed of two transcription factors (TF). Typically, one TF is a developmentally regulated factor that is essential for proper specification of a given lineage and provides the DNA-binding domain, while the partner TF contributes a transactivation domain that drives aberrant expression of target genes. Based on these common genetic characteristics, the first ESF-EMBO research conference entitled “Molecular Biology and Innovative Therapies in Sarcomas of Childhood and Adolescence” with special focus on RMS and ES was held at the Polonia Castle in Pultusk, Poland. The conference gathered 70 participants from more than 15 countries and several continents representing most research groups that are active in this field.
Inhibition of the proteasome is considered as a promising strategy to sensitize cancer cells to apoptosis. Recently, we demonstrated that the proteasome inhibitor Bortezomib primes neuroblastoma cells to TRAIL-induced apoptosis. In the present study, we investigated whether Bortezomib increases chemosensitivity of neuroblastoma cells. Unexpectedly, we discover an antagonistic interaction of Bortezomib and microtubule-interfering drugs. Bortezomib significantly attenuates the loss of cell viability and induction of apoptosis on treatment with Taxol and different vinca alkaloids but not with other chemotherapeutics, that is, Doxorubicin and Cisplatinum. Importantly, Bortezomib inhibits G2/M transition by inhibiting proteasomal degradation of cell cycle regulatory proteins such as p21, thereby preventing cells to enter mitosis, the cell cycle phase in which they are most vulnerable to antitubulin chemotherapeutics. Consequently, Bortezomib counteracts Taxol-induced mitotic arrest and polyploidy, as shown by reduced expression of PLK1 and phosphorylated histone H3. In addition, Bortezomib antagonizes Taxol-mediated degradation of MCL-1 during mitotic arrest by preventing cells to enter mitosis and by inhibiting the proteasome. Downregulation of MCL-1 is critically required for Taxol-induced apoptosis, as overexpression of a phosphomutant MCL-1 variant, which is resistant to degradation, significantly diminishes Taxol-triggered apoptosis. Vice versa, attenuation of Bortezomib-mediated accumulation of MCL-1 by knockdown of MCL-1 significantly enhances Taxol/Bortezomib-induced apoptosis. Thus, Bortezomib rescues Taxol-induced apoptosis by inhibiting G2/M transition and mitigating MCL-1 degradation. The identification of this antagonistic interaction of Bortezomib and microtubule-targeted drugs has important implications for the design of Bortezomib-based combination therapies.
Since most anticancer therapies including immunotherapy trigger programmed cell death in cancer cells, defective cell death programs can lead to treatment resistance and tumor immune escape. Therefore, evasion of programmed cell death may provide one possible explanation as to why cancer immunotherapy has so far only shown modest clinical benefits for children with cancer. A better understanding of the molecular mechanisms that regulate sensitivity and resistance to programmed cell death is expected to open new perspectives for the development of novel experimental treatment strategies to enhance the efficacy of cancer immunotherapy in the future.
While necroptosis has for long been viewed as an accidental mode of cell death triggered by physical or chemical damage, it has become clear over the last years that necroptosis can also represent a programmed form of cell death in mammalian cells. Key discoveries in the field of cell death research, including the identification of critical components of the necroptotic machinery, led to a revised concept of cell death signaling programs. Several regulatory check and balances are in place in order to ensure that necroptosis is tightly controlled according to environmental cues and cellular needs. This network of regulatory mechanisms includes metabolic pathways, especially those linked to mitochondrial signaling events. A better understanding of these signal transduction mechanisms will likely contribute to open new avenues to exploit our knowledge on the regulation of necroptosis signaling for therapeutic application in the treatment of human diseases.