Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.
Dendritic cells (DCs) occupy a privileged position at the interface between innate and adaptive immunity, orchestrating a large panel of responses to both physiological and pathological cues. In particular, whereas the presentation of antigens by immature DCs generally results in the development of immunological tolerance, mature DCs are capable of priming robust, and hence therapeutically relevant, adaptive immune responses. In line with this notion, functional defects in the DC compartment have been shown to etiologically contribute to pathological conditions including (but perhaps not limited to) infectious diseases, allergic and autoimmune disorders, graft rejection and cancer. Thus, the possibility of harnessing the elevated immunological potential of DCs for anticancer therapy has attracted considerable interest from both researchers and clinicians over the last decade. Alongside, several methods have been developed not only to isolate DCs from cancer patients, expand them, load them with tumor-associated antigens and hence generate highly immunogenic clinical grade infusion products, but also to directly target DCs in vivo. This intense experimental effort has culminated in 2010 with the approval by the US FDA of a DC-based preparation (sipuleucel-T, Provenge®) for the treatment of asymptomatic or minimally symptomatic metastatic castration-refractory prostate cancer. As an update to the latest Trial Watch dealing with this exciting field of research (October 2012), here we summarize recent advances in DC-based anticancer regimens, covering both high-impact studies that have been published during the last 13 mo and clinical trials that have been launched in the same period to assess the antineoplastic potential of this variant of cellular immunotherapy.
antigen-presenting cells; immunogenic cell death; monoclonal antibodies; plasmacytoid dendritic cells; regulatory T cells; Toll-like receptors
Radiotherapy has extensively been employed as a curative or palliative intervention against cancer throughout the last century, with a varying degree of success. For a long time, the antineoplastic activity of X- and γ-rays was entirely ascribed to their capacity of damaging macromolecules, in particular DNA, and hence triggering the (apoptotic) demise of malignant cells. However, accumulating evidence indicates that (at least part of) the clinical potential of radiotherapy stems from cancer cell-extrinsic mechanisms, including the normalization of tumor vasculature as well as short- and long-range bystander effects. Local bystander effects involve either the direct transmission of lethal signals between cells connected by gap junctions or the production of diffusible cytotoxic mediators, including reactive oxygen species, nitric oxide and cytokines. Conversely, long-range bystander effects, also known as out-of-field or abscopal effects, presumably reflect the elicitation of tumor-specific adaptive immune responses. Ionizing rays have indeed been shown to promote the immunogenic demise of malignant cells, a process that relies on the spatiotemporally defined emanation of specific damage-associated molecular patterns (DAMPs). Thus, irradiation reportedly improves the clinical efficacy of other treatment modalities such as surgery (both in neo-adjuvant and adjuvant settings) or chemotherapy. Moreover, at least under some circumstances, radiotherapy may potentiate anticancer immune responses as elicited by various immunotherapeutic agents, including (but presumably not limited to) immunomodulatory monoclonal antibodies, cancer-specific vaccines, dendritic cell-based interventions and Toll-like receptor agonists. Here, we review the rationale of using radiotherapy, alone or combined with immunomodulatory agents, as a means to elicit or boost anticancer immune responses, and present recent clinical trials investigating the therapeutic potential of this approach in cancer patients.
brachytherapy; immunogenic cell death; intensity-modulated radiation therapy; radionuclide; stereotactic body radiation therapy; stereotactic radiosurgery
The illicit generation of tetraploid cells constitutes a prominent driver of oncogenesis, as it often precedes the development of aneuploidy and genomic instability. In addition, tetraploid (pre-)malignant cells display an elevated resistance against radio- and chemotherapy. Here, we report a strategy to preferentially kill tetraploid tumor cells based on the broad-spectrum kinase inhibitor SP600125. Live videomicroscopy revealed that SP600125 affects the execution of mitosis, impedes proper cell division and/or activates apoptosis in near-to-tetraploid, though less so in parental, cancer cells. We propose a novel graphical model to quantify the differential response of diploid and tetraploid cells to mitotic perturbators, including SP600125, which we baptized “transgenerational cell fate profiling.” We speculate that this representation constitutes a valid alternative to classical “single-cell fate” and “genealogical” profiling and, hence, may facilitate the analysis of cell fate within a heterogeneous population as well as the visual examination of cell cycle alterations.
cell death; cytokinesis failure; mitotic catastrophe; microtubules; polyploidy; time-lapse microscopy
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
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
For a long time, it was commonly believed that efficient anticancer regimens would either trigger the apoptotic demise of tumor cells or induce a permanent arrest in the G1 phase of the cell cycle, i.e., senescence. The recent discovery that necrosis can occur in a regulated fashion and the increasingly more precise characterization of the underlying molecular mechanisms have raised great interest, as non-apoptotic pathways might be instrumental to circumvent the resistance of cancer cells to conventional, pro-apoptotic therapeutic regimens. Moreover, it has been shown that some anticancer regimens engage lethal signaling cascades that can ignite multiple oncosuppressive mechanisms, including apoptosis, necrosis, and senescence. Among these signaling pathways is mitotic catastrophe, whose role as a bona fide cell death mechanism has recently been reconsidered. Thus, anticancer regimens get ever more sophisticated, and often distinct strategies are combined to maximize efficacy and minimize side effects. In this review, we will discuss the importance of apoptosis, necrosis, and mitotic catastrophe in the response of tumor cells to the most common clinically employed and experimental anticancer agents.
caspases; lysosomal membrane permeabilization; mitochondrial membrane permeabilization; necrosome; oncosis; phosphatidylserine; RIP1; reactive oxygen species
In the last 20 years, the field of cellular and molecular oncology has been born and has moved its first steps, with an increasingly rapid pace. Hundreds of oncogenic and oncosuppressive signaling cascades have been characterized, facilitating the development of an ever more refined and variegated arsenal of diagnostic and therapeutic weapons. Furthermore, several cancer-specific features and processes have been identified that constitute promising therapeutic targets. For instance, it has been demonstrated that microRNAs can play a critical role in oncogenesis and tumor suppression. Moreover, it turned out that tumor cells frequently exhibit an extensive metabolic rewiring, can behave in a stem cell-like fashion (and hence sustain tumor growth), often constitutively activate stress response pathways that allow them to survive, can react to therapy by engaging in non-apoptotic cell death programs, and sometimes die while eliciting a tumor-specific immune response. In this Perspective article, we discuss the main issues generated by these discoveries that will be in the limelight of molecular and cellular oncology research for the next, hopefully few years.
immunogenic cell death; necroptosis; non-oncogene addiction; regulated necrosis; oncometabolites
Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that knockout, knockdown or pharmacological inhibition of p53 can induce autophagy in human, mouse and nematode cells. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53-/- cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.
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.