The antineoplastic agent cis-diammineplatinum(II) dichloride (cisplatin, CDDP) is part of the poorly effective standard treatment of non-small cell lung carcinoma (NSCLC). Here, we report a novel strategy to improve the efficacy of CDDP. In conditions in which CDDP alone or either of two PARP inhibitors, PJ34 hydrochloride hydrate or CEP 8983, used as standalone treatments were inefficient in killing NSCLC cells, the combination of CDDP plus PJ34 or that of CDDP plus CEP 8983 were found to kill a substantial fraction of the cells. This cytotoxic synergy could be recapitulated by combining CDDP and the siRNA-mediated depletion of the principal PARP isoform, PARP1, indicating that it is mediated by on-target effects of PJ34 or CEP 8983. CDDP and PARP inhibitors synergized in inducing DNA damage foci, mitochondrial membrane permeabilization leading to cytochrome c release, and dissipation of the inner transmembrane potential, caspase activation, plasma membrane rupture and loss of clonogenic potential in NSCLC cells. Collectively, our results indicate that CDDP can be advantageously combined with PARP inhibitors to kill several NSCLC cell lines, independently from their p53 status. Combined treatment with CDDP and PARP inhibitors elicits the intrinsic pathway of apoptosis.
apoptosis; CEP 8983; DNA damage response; A549 cells; PJ34 hydrochloride; small-interfering RNA
A chemical screen designed to identify novel inducers of autophagy led to the discovery that signal transducer and activator of transcription 3 (STAT3) inhibitors can potently stimulate the autophagic flux. Although STAT3 is best known as a pro-inflammatory and oncogenic transcription factor, mechanistic analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear, pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase 2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA. This interaction, which could be recapitulated using recombinant proteins in pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus, STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e., Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter does so in cell-free systems in vitro. A further screen designed to identify EIF2AK2-dependent autophagy inducers revealed that several fatty acids including palmitate trigger autophagy via a pathway that involves the disruption of the STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These results reveal an unsuspected crosstalk between cellular metabolism (fatty acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and translational control (EIF2S1) that regulates autophagy.
EIF2S1S51A; endoplasmic reticulum; IRS1; palmitate; polyI:C; STAT3Y705F
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
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
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
Background. In folk medicine, extra virgin olive oil (EVOO) is used as a remedy for a variety of diseases. This study investigates the in vivo antinociceptive, anti-inflammatory, and anti-cancer effects of EVOO on mice and rats. Materials and Methods. In this experimental study, using the acetic acid-induced writhing and formalin tests in mice, the analgesic effect of EVOO was evaluated. Acetylsalicylic acid and morphine were used as standard drugs, respectively. The anti-inflammatory activity was investigated by means of the carrageenan-induced paw edema model in rats using acetylsalicylic acid and dexamethasone as standard drugs. Last, the xenograft model in athymic mice was used to evaluate the anticancer effect in vivo. Results. EVOO significantly decreased acetic acid-induced abdominal writhes and reduces acute and inflammatory pain in the two phases of the formalin test. It has also a better effect than Dexamethasone in the anti-inflammatory test. Finally, the intraperitoneal administration of EVOO affects the growth of HCT 116 tumours xenografted in athymic mice. Conclusion. EVOO has a significant analgesic, anti-inflammatory, and anticancer properties. However, further detailed studies are required to determine the active component responsible for these effects and mechanism pathway.
During the past two decades, several interventions have been shown to increase the healthy lifespan of model organisms as evolutionarily distant from each other as yeast, worms, flies and mammals. These anti-aging maneuvers include (but are not limited to) cycles of caloric restriction, physical exercise as well as the administration of multiple, chemically unrelated agents, such as resveratrol, spermidine and various rapamycin-like compounds collectively known as rapalogs. Most, if not all, lifespan-extending agents promote macroautophagy (hereafter referred to as autophagy), an evolutionarily old mechanism that contributes to the maintenance of intracellular homeostasis and plays a critical role in the adaptive response of cells to stress. In line with this notion, the activation of autophagy appears to mediate significant anti-ageing effects in several organisms, including mice. Here, we focus on rapalogs to discuss the possibility that part of the beneficial activity of lifespan-extending agents stems from their ability to exert immunostimulatory effects. Accumulating evidence indicates indeed that the immune system can recognize and eliminate not only cells that are prone to undergo malignant transformation, but also senescent cells, thus playing a significant role in the control of organismal aging. In addition, it has recently become clear that rapamycin and other rapalogs, which for a long time have been viewed (and used in the clinic) as pure immunosuppressants, can mediate robust immunostimulatory functions, at least in some circumstances.
ATG5; everolimus; mTOR; sirolimus; tacrolimus; temsirolimus
The foundation of modern vaccinology dates back to the 1790s, when the English physician Edward Jenner uncovered the tremendous medical potential of prophylactic vaccination. Jenner’s work ignited a wave of nationwide vaccination campaigns abating the incidence of multiple life-threatening infectious diseases and culminating with the eradication of natural smallpox virus, which was definitively certified by the WHO in 1980. The possibility of using vaccines against cancer was first proposed at the end of the 19th century by Paul Ehrlich and William Coley. However, it was not until the 1990s that such a hypothesis began to be intensively investigated, following the realization that the immune system is not completely unresponsive to tumors and that neoplastic cells express immunogenic tumor-associated antigens (TAAs). Nowadays, anticancer vaccines are rapidly moving from the bench to the bedside, and a few prophylactic and therapeutic preparations have already been approved by FDA for use in humans. In this setting, one interesting approach is constituted by DNA vaccines, i.e., TAA-encoding circularized DNA constructs, often of bacterial origin, that are delivered to patients as such or by means of specific vectors, including (but not limited to) liposomal preparations, nanoparticles, bacteria and viruses. The administration of DNA vaccines is most often performed via the intramuscular or subcutaneous route and is expected to cause (1) the endogenous synthesis of the TAA by myocytes and/or resident antigen-presenting cells; (2) the presentation of TAA-derived peptides on the cell surface, in association with MHC class I molecules; and (3) the activation of potentially therapeutic tumor-specific immune responses. In this Trial Watch, we will summarize the results of recent clinical trials that have evaluated/are evaluating DNA vaccines as therapeutic interventions against cancer.
cross-presentation; dendritic cells; electroporation; mucosal immunity; Saccharomyces cerevisiae; Salmonella typhimurium
It is now clear that the immune system plays a critical role not only during oncogenesis and tumor progression, but also as established neoplastic lesions respond to therapy. Selected cytotoxic chemicals can indeed elicit immunogenic cell death, a functionally peculiar type of apoptosis that stimulates tumor-specific cognate immune responses. Such immunogenic chemotherapeutics include cyclophosphamide, doxorubicin and oxaliplatin (which are approved by FDA for the treatment of various hematological and solid malignancies), mitoxantrone (which is currently employed both as an anticancer agent and against multiple sclerosis) and patupilone (a microtubular poison in clinical development). One year ago, in the second issue of OncoImmunology, we discussed the scientific rationale behind immunogenic chemotherapy and reviewed the status of recent clinical trials investigating the off-label use of cyclophosphamide, doxorubicin, oxaliplatin and mitoxantrone in cancer patients. Here, we summarize the latest developments in this area of clinical research, covering both high-impact studies that have been published during the last 13 months and clinical trials that have been initiated in the same period to assess the antineoplastic profile of immunogenic chemotherapeutics.
autophagy; ATP; calreticulin; dendritic cells; epothilone B; HMGB1
The BCL-2 homolog BCL-XL, one of the two protein products of BCL2L1, has originally been characterized for its prominent prosurvival functions. Similar to BCL-2, BCL-XL binds to its multidomain proapoptotic counterparts BAX and BAK, hence preventing the formation of lethal pores in the mitochondrial outer membrane, as well as to multiple BH3-only proteins, thus interrupting apical proapoptotic signals. In addition, BCL-XL has been suggested to exert cytoprotective functions by sequestering a cytosolic pool of the pro-apoptotic transcription factor p53 and by binding to the voltage-dependent anion channel 1 (VDAC1), thereby inhibiting the so-called mitochondrial permeability transition (MPT). Thus, BCL-XL appears to play a prominent role in the regulation of multiple distinct types of cell death, including apoptosis and regulated necrosis. More recently, great attention has been given to the cell death-unrelated functions of BCL-2-like proteins. In particular, BCL-XL has been shown to modulate a number of pathophysiological processes, including—but not limited to—mitochondrial ATP synthesis, protein acetylation, autophagy and mitosis. In this short review article, we will discuss the functions of BCL-XL at the interface between cell death and metabolism.
One of the driving forces of oncogenesis is tetraploidy, a duplication of the DNA content that, upon asymmetric cell division or progressive chromosome loss, can originate aneuploidy. Recent findings from our group indicate the existence of an immunosurveillance system that eliminates tetraploid cancer cells. We surmise that tetraploidy-inducing chemotherapeutic agents may elicit potent anticancer responses by re-activating this immunosurveillance system.
breast carcinoma; calreticulin; HMGB1; hyperploidy; immunogenic cell death; mitotic catastrophe
Solid tumors are constituted of a variety of cellular components, including bona fide malignant cells as well as endothelial, structural and immune cells. On one hand, the tumor stroma exerts major pro-tumorigenic and immunosuppressive functions, reflecting the capacity of cancer cells to shape the microenvironment to satisfy their own metabolic and immunological needs. On the other hand, there is a component of tumor-infiltrating leucocytes (TILs) that has been specifically recruited in the attempt to control tumor growth. Along with the recognition of the critical role played by the immune system in oncogenesis, tumor progression and response to therapy, increasing attention has been attracted by the potential prognostic and/or predictive role of the immune infiltrate in this setting. Data from large clinical studies demonstrate indeed that a robust infiltration of neoplastic lesions by specific immune cell populations, including (but not limited to) CD8+ cytotoxic T lymphocytes, Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages constitutes an independent prognostic indicator in several types of cancer. Conversely, high levels of intratumoral CD4+CD25+FOXP3+ regulatory T cells, Th2 CD4+ T cells, myeloid-derived suppressor cells, M2 macrophages and neutrophils have frequently been associated with dismal prognosis. So far, only a few studies have addressed the true predictive potential of TILs in cancer patients, generally comforting the notion that—at least in some clinical settings—the immune infiltrate can reliably predict if a specific patient will respond to therapy or not. In this Trial Watch, we will summarize the results of clinical trials that have evaluated/are evaluating the prognostic and predictive value of the immune infiltrate in the context of solid malignancies.
biomarker; chemotherapy; cytokines; interferon γ; interleukin-10; plasmacytoid dendritic cells; transforming growth factor
Dendritic cells (DCs) occupy a central position in the immune system, orchestrating a wide repertoire of responses that span from the development of self-tolerance to the elicitation of potent cellular and humoral immunity. Accordingly, DCs are involved in the etiology of conditions as diverse as infectious diseases, allergic and autoimmune disorders, graft rejection and cancer. During the last decade, several methods have been developed to load DCs with tumor-associated antigens, ex vivo or in vivo, in the attempt to use them as therapeutic anticancer vaccines that would elicit clinically relevant immune responses. While this has not always been the case, several clinical studies have demonstrated that DC-based anticancer vaccines are capable of activating tumor-specific immune responses that increase overall survival, at least in a subset of patients. In 2010, this branch of clinical research has culminated with the approval by FDA of a DC-based therapeutic vaccine (sipuleucel-T, Provenge®) for use in patients with asymptomatic or minimally symptomatic metastatic hormone-refractory prostate cancer. Intense research efforts are currently dedicated to the identification of the immunological features of patients that best respond to DC-based anticancer vaccines. This knowledge may indeed lead to personalized combination strategies that would extend the benefit of DC-based immunotherapy to a larger patient population. In addition, widespread enthusiasm has been generated by the results of the first clinical trials based on in vivo DC targeting, an approach that holds great promises for the future of DC-based immunotherapy. In this Trial Watch, we will summarize the results of recently completed clinical trials and discuss the progress of ongoing studies that have evaluated/are evaluating DC-based interventions for cancer therapy.
CD8+ cytotoxic T lymphocytes; Provenge®; Toll-like receptors; antigen-presenting cells; immunotherapy; pulsed dendritic cells
Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part “private,” i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance
N-acetyl-cysteine; autophagy; bongkrekic acid; cyclosporine A; glutathione; large-amplitude swelling
aneuploidy; depolyploidization; tetraploidy; oncogenesis; polyploidy
The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53+/+ and TP53−/− human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53−/− (but not TP53+/+) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53−/− cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53−/− cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.
caspases; HCT 116; high-throughput screening; mitochondrial outer membrane permeabilization; MPS1
Tetraploidy constitutes an adaptation to stress and an intermediate step between euploidy and aneuploidy in oncogenesis. Tetraploid cells are particularly resistant against genotoxic stress including radiotherapy and chemotherapy. Here, we designed a strategy to preferentially kill tetraploid tumor cells. Depletion of checkpoint kinase-1 (Chk1) by siRNAs, transfection with dominant-negative Chk1 mutants or pharmacological Chk1 inhibition killed tetraploid colon cancer cells yet had minor effects on their diploid counterparts. Chk1 inhibition abolished the spindle assembly checkpoint and caused premature and abnormal mitoses that led to p53 activation and cell death at a higher frequency in tetraploid than in diploid cells. Similarly, abolition of the spindle checkpoint by knockdown of Bub1, BubR1 or Mad2 induced p53-dependent apoptosis of tetraploid cells. Chk1 inhibition reversed the cisplatin resistance of tetraploid cells in vitro and in vivo, in xenografted human cancers. Chk1 inhibition activated p53-regulated transcripts including Puma/BBC3 in tetraploid but not in diploid tumor cells. Altogether, our results demonstrate that, in tetraploid tumor cells, the inhibition of Chk1 sequentially triggers aberrant mitosis, p53 activation and Puma/BBC3-dependent mitochondrial apoptosis.