Loss of anchorage primarily induces non-apoptotic cell death in a human mammary epithelial cell line under atypical focal adhesion kinase signaling

Anchorage dependence of cellular growth and survival prevents inappropriate cell growth or survival in ectopic environments, and serves as a potential barrier to metastasis of cancer cells. Therefore, obtaining a better understanding of anchorage-dependent responses in normal cells is the first step to understand and impede anchorage independence of growth and survival in cancer cells and finally to eradicate cancer cells during metastasis. Anoikis, a type of apoptosis specifically induced by lack of appropriate cell-extracellular matrix adhesion, has been established as the dominant response of normal epithelial cells to anchorage loss. For example, under detached conditions, the untransformed mammary epithelial cell (MEC) line MCF-10 A, which exhibits myoepithelial characteristics, underwent anoikis dependent on classical ERK signaling. On the other hand, recent studies have revealed a variety of phenotypes resulting in cell death modalities distinct from anoikis, such as autophagy, necrosis, and cornification, in detached epithelial cells. In the present study, we characterized detachment-induced cell death (DICD) in primary human MECs immortalized with hTERT (^TertHMECs), which are bipotent progenitor-like cells with a differentiating phenotype to luminal cells. In contrast to MCF-10 A cells, apoptosis was not observed in detached ^TertHMECs; instead, non-apoptotic cell death marked by features of entosis, cornification, and necrosis was observed along with downregulation of focal adhesion kinase (FAK) signaling. Cell death was overcome by anchorage-independent activities of FAK but not PI3K/AKT, SRC, and MEK/ERK, suggesting critical roles of atypical FAK signaling pathways in the regulation of non-apoptotic cell death. Further analysis revealed an important role of TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) as a mediator of FAK signaling in regulation of entosis and necrosis and a role of p38 MAPK in the induction of necrosis. Overall, the present study highlighted outstanding cell subtype or differentiation stage specificity in cell death phenotypes induced upon anchorage loss in human MECs.Normal cells undergo cell death and/or growth arrest in the absence of attachment to extracellular matrix (ECM) or upon contact with abnormal or ectopic ECM, which constitutes a physiologically important defense mechanism in multicellular organisms for preventing re-adhesion of detached cells to foreign matrices and their dysplastic growth in inappropriate sites.^{1, 2} On the other hand, the process of cancer metastasis demands that cancer cells circumvent such cell death/growth arrest. This is true even for incipient tumors, where outgrowth and displacement of cells from their original location in a mass result in loss of adequate contact of cells with innate ECM. Cells that disseminate through foreign stroma experience more deviant conditions, and upon reaching the parenchyma of distant organs need to adapt to the non-permissive matrix in the foreign tissue. To survive through this process, cancer cells acquire resistance to cell death/growth arrest induced in the absence of appropriate adhesion to ECM. Therefore, the eradication of cancer cells in ectopic environments requires an understanding of their resistance to anchorage dependence for growth and survival based on responsiveness of their normal counterparts.Anoikis is a particular type of apoptosis that is induced by inadequate or inappropriate cell–ECM interactions, and is the best-characterized phenotype induced by loss of anchorage in anchorage-dependent epithelial cells.^{2, 3} On the other hand, detachment of cells from ECM has been observed to induce a variety of cell death phenotypes that are distinct from the typical anoikis; these include entosis, autophagy, and squamous transdifferentiation.^{4, 5, 6, 7, 8} The emerging diversity of cell death phenotypes necessitates extension of the study of adhesion-dependent cell death beyond classical anoikis.A considerable number of studies have suggested that anoikis is the predominant cell death phenotype induced in mammary epithelial cells (MECs) upon anchorage loss;^{9, 10, 11, 12, 13} however, many of these studies employed rodent cells or the human cell line MCF-10 A, which has been characterized as being predominantly myoepithelial or classified into basal B subtype.^{14, 15, 16} Given that the majority of malignant breast cancers exhibit the luminal characteristics, a phenotype based on a normal counterpart or a correspondent luminal subtype of human MECs needs to be defined, particularly given the current limited knowledge in this respect.In the present study, we characterized anchorage loss-induced cell death in MECs using primary human MECs immortalized with hTERT (^TertHMEC).^{17, 18} The established cells are potential stem/progenitors of mammary epithelial cells¹⁸ and show a partial differentiation toward to the luminal phenotype in the culture system developed by Stampfer et al (http://hmec.lbl.gov/mreview.htm). Unlike previous observations based on MCF-10 A cells, the detached ^TertHMECs were found to have an apparent defect in the execution of apoptosis and instead, underwent non-apoptotic cell death through simultaneous entosis, cornification, and necrotic processes. The roles of focal adhesion kinase (FAK) and its atypical signaling mediated by TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand) in this process have been highlighted.     

Synergistic anti-tumor activity and inhibition of angiogenesis by cotargeting of oncogenic and death receptor pathways in human melanoma

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244–TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244–TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFβ1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.The development of mutant BRAF (v-raf murine sarcoma viral oncogene homolog B)- and mitogen/extracellular signal-regulated kinase (MEK)-specific inhibitors, such as Vemurafenib, Dabrafenib and Trametinib, as well as of monoclonal antibodies targeting immune checkpoints, has markedly improved the treatment of advanced melanoma, as shown by highly significant effects, achieved in several trials, on progression-free and/or overall survival.^{1, 2, 3, 4, 5} However, a fraction of patients does not benefit from target-specific therapy or immunotherapy, and duration of clinical responses may be limited.^{1, 2, 3, 4, 5} Mechanisms of resistance to specific inhibitors⁶ and of tumor escape from immune recognition⁷ contribute to prevent induction of melanoma cell death by the new therapies and explain the urgent need for the identification of more effective approaches. Different strategies are being investigated to overcome melanoma resistance to single anti-tumor agents and to rescue tumor susceptibility to cell death, including co-targeting of constitutively active intracellular signaling pathways,^{8, 9, 10} association of target-specific drugs with inhibitors of autophagy or with endoplasmic reticulum-stress inducers^11,12 and association of anti-tumor agents that trigger the extrinsic and the intrinsic pathway of apoptosis.^{13, 14, 15}The latter approach is based on the combination of specific inhibitors of main oncogenic pathways, which in different tumor types can modulate relevant pro- and anti-apoptotic molecules in the intrinsic pathway of cell death,^{16, 17, 18} with targeting of the extrinsic, death receptor-dependent pathway, by usage of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or of agonistic death receptor 5 (DR5)-specific mAbs.¹⁹ Indeed, this approach has shown that association of MEK, pan-RAF or phosphoinositide 3-kinase (PI3K) inhibitors with TRAIL can overcome resistance to TRAIL^{13, 14, 15} and can lead to enhanced melanoma apoptosis in vitro through different mechanisms, including upregulation of bcl-2-like protein 11 isoform 1 (Bim) and activation of BCL2-associated X protein (Bax).^{13, 14, 15} Moreover, as hypothesized recently by Geserick et al.,²⁰ the association of MEK or pan-RAF inhibitors with TRAIL could even be exploited as a potential approach to promote rapid elimination of most tumor cells, thus preventing the emergence of secondary resistance to BRAF inhibitors. Furthermore, the interest in the death receptor pathway, as a therapeutic target, has been recently strengthened by the evidence that TRAIL mediates disruption of the tumor-associated vasculature²¹ and by the discovery of TIC10, a drug that stimulates production of TRAIL and that exerts significant anti-tumor activities in preclinical in vivo models, including aggressive intracranial xenografts of human glioblastoma cells.²²Nevertheless, it is currently not known whether co-targeting of MEK and/or PI3K/mammalian target of rapamycin (mTOR) and of the death receptor pathway in melanoma can overcome intrinsic resistance to each of the anti-tumor agents in most instances, irrespective of the different genetic make-up of the tumors, and whether this approach can exert synergistic, rather than additive, anti-melanoma effects. Furthermore, it remains to be verified whether the combination of MEK or PI3K/mTOR inhibitors with death receptor agonists (such as TRAIL itself or DR5-specific mAbs) may also exert significant pro-apoptotic effects in vivo on melanoma xenografts and whether this is associated with inhibition of relevant pro-tumoral processes in the tumor microenvironment.To address these issues, in this study we evaluated the anti-melanoma activity in vitro and in vivo of two- or three-drug associations using TRAIL, the MEK 1/2 inhibitor AZD6244/Selumetinib, which has significant clinical activity in melanoma,²³ and the PI3K/mTOR inhibitor BEZ235, currently in clinical trials in different solid tumors, including melanoma (source www.clinicaltrials.gov). The results indicated that the three-agent (AZD6244/BEZ235/TRAIL) and two-agent (AZD6244/TRAIL) combinations exerted synergistic pro-apoptotic effects on most melanomas in a large panel. These results were observed even on melanoma cell lines resistant to TRAIL or to the inhibitors and independently of their BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS), p53 and phosphatase and tensin homolog (PTEN) status. Moreover, an in vivo model showed that the AZD6244/TRAIL association promoted melanoma apoptosis associated with marked inhibition of angiogenesis.     

Entosis,a key player in cancer cell competition

Cell-in-cell structures, also referred to as ''entosis'', are frequently found in human malignancies, although their prognostic impact remains to be defined. Two articles recently published in Cell Research report the stimulation of entosis by one prominent oncogene, Kras, as well as by one class of tumor suppressors, namely epithelial cadherins E and P, illustrating the complex regulation of this biological process.A number of different terms have been used to describe live cell engulfments giving rise to cell-in-cell structures (CICS): entosis, emperipolesis, cannibalism and phagocytosis. Heterotypic live cell engulfment usually involves the ingestion of leukocytes by non-leukocytes (such as epithelial cells or fibroblasts). Homotypic live cell engulfment (among cells of the same type) mostly occurs in cancers, probably reflecting major alterations in cellular physiology that are associated with oncogenesis and tumor progression.CICS can be visualized by conventional hematoxylin-eosin staining and have been described to occur in many different human cancers¹. CICS produced as the result of entosis exhibit β-catenin localization patterns that are indicative of a cell junction-mediated mechanism of engulfment, and this polarized distribution of β-catenin can be taken advantage of to visualize CICS in vivo, in tumors¹. However, the prognostic impact of CICS is highly context-dependent. Thus, CICS are particularly frequent in high-grade, aggressive breast cancer with dismal prognosis². CICS are only found in castration-resistant, not in androgen-dependent, prostate cancer and hence correlate with poor prognosis in this particular malignancy³. In contrast, in pancreas adenocarcinomas, high levels of CICS correlate with a lower incidence of metastases⁴. These findings point to a complex role of CICS in cancer biology.Two papers by Sun et al.^5,6 recently published in Cell Research characterized one particular mechanism of homotypic live cell engulfment termed entosis. The first paper of this series⁵ provides evidence that one of the most prominent oncogenes, activated Kras, can stimulate entosis, while the second paper⁶ demonstrates that a prominent tumor suppressor, epithelial cadherin (E-cadherin), can increase entosis as well.Cancers are highly complex mixtures of cells in which the malignant population is genetically and epigenetically heterogeneous, reflecting a history of clonal selection. One particular type of competition among distinct cells may consist in the engulfment of one cell (the ''loser'') by another (the ''winner''), as demonstrated by Sun et al. in several cell culture models, as well as in human cancers that were xenografted into immunodeficient mice⁵. Importantly, co-culture of non-transformed cells with their malignant counterparts systematically leads to engulfment of the former by the latter, suggesting that oncogenic transformation is coupled to the ''winner'' status⁵. Indeed, competition by entosis leads to the physical elimination of the ''loser'' cells, which usually succumb to non-apoptotic cell death as soon as the phagosome enveloping the engulfed cell is decorated with LC3 and then fuses with lysosomes^1,7. What is then the difference between ''loser'' and ''winner'' cells? Sun et al.⁵ propose that one cardinal feature of ''winners'' is a high degree of mechanic deformability, as demonstrated by biophysical experiments and computer simulations. This is a highly provocative finding because human tumors are known to be more mechanically heterogeneous than normal tissues and that tumor progression is increased with an elevated mechanic deformability of the cancer cells. This reduction in cell stiffness may hence not only increase the metastatic potential of tumor cells⁸, but may also reflect an increased entotic activity⁵.Transfection-enforced expression of active KrasV12 was sufficient to confer winner status onto non-tumorigenic cells, correlating with an increase in mechanic deformability⁵. This effect of KrasV12 relied on Rac1, as demonstrated by the facts that knockdown of Rac1 suppressed the ''winner'' status conferred by KrasV12, expression of constitutively active Rac1 induced a ''winner'' phenotype and dominant-negative Rac1N17 imparted a ''loser'' status⁵. However, at this point it remains to be explored whether other pathways downstream of Kras such as the phosphatidylinositide 3-kinases (PI3K)/protein kinase B (PKB, best known as AKT)/mechanistic target of rapamycin (mTOR) pathway may contribute to ''winner'' status. Inhibition of mTOR interferes with degradation of engulfed cells⁹, suggesting that activation of the PI3K/AKT/mTOR axis might favor the manifestation of the ''winner'' phenotype as well. Similarly, it remains an open question as to whether other oncogenes than Kras may regulate entosis as well.Breast cancers cells engineered to express epithelal E- or P-cadherins (but not mesenchymal-type cadherins, such as N-cadherin and cadherin-11) re-establish epithelial junctions and engulf and kill non-transfected parental cells in transformed growth assays⁶. The induction of entosis by epithelial E- or P-cadherins is associated to the polarized distribution of RhoA and contractile actomyosin dependent on the p190A Rho-GTPase-Activating Protein (p190A RhoGAP) that is recruited to epithelial junctions⁶. Inhibition of RhoA by overexpression of RhoA-N19 or p190A RhoGAP was sufficient to impart winner status to cells mixed with controls, whereas overexpression of RhoA, ROCKI, or ROCKII had the opposite effect and hence created ''loser'' cells⁵. It has been known that Rho-GTPase and Rho-kinase are not required in engulfing cells but are required in internalizing cells¹, underscoring the idea that ''loser'' cells are not just passive ''victims'' of a cannibalistic attack but somehow contribute to their fatal fate. The ''loser'' status was accompanied by the ROCK-dependent accumulation of actomyosin⁶, and computer simulations suggest that actomyosin contractility within ''loser'' cells constitutes a critical driving force of entosis⁵. The levels of phosphorylated myosin light chain 2 at Ser19 (pMLC2), a readout of contractile myosin downstream of ROCKI/II, were also increased in ''loser'' cells as compared to ''winners''⁶. RhoA, ROCKI/II, MLC2, actin and myosins all accumulated at particularly high levels in ''losers'' at the cell cortex oriented away from cell-cell adhesions⁶.The aforementioned data support a dual implication of entosis in carcinogenesis (Figure 1). On one hand, entosis carried out by ''winner'' cells may constitute a competitive advantage of aggressive tumor cells, perhaps allowing the ''winners'' to retrieve amino acids and other building blocks for anabolic reaction from their cannibalistic activity⁹ or increasing their genomic instability subsequent to mitotic aberrations^2,10. In this context, pharmacological suppression of entosis by Y27632, a ROCKI/II inhibitor, abolished the competitive advantage of transformed cells over their non-transformed siblings in mixed culture experiments⁵. On the other hand, stimulation of entosis by re-expression of epithelal E- or P-cadherins reduced the clonogenic potential of breast cancer cells. In this context, Y27632 facilitated tumor cell growth in vitro⁶. These observations underscore the need of exploring the detailed mechanisms through which entosis may repress or favor oncogenesis and tumor progression.Open in a separate windowFigure 1A dual role for entosis in cancer. (A) Entosis as a pro-tumorigenic process. (B) Entosis as a tumor-suppressive mechanism.     

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent,E6-independent mechanism

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common cancer worldwide.¹ While the overall incidence of HNSCC, traditionally associated with tobacco or alcohol consumption, is declining, a subset of oropharyngeal cancers caused by infection with high-risk types of human papillomavirus (HPV) has risen significantly.^2,3 Transformation upon HPV infection occurs mainly because of inactivation of the p53 and retinoblastoma tumour suppressor proteins mediated by the viral oncoproteins E6 and E7, respectively.⁴HPV-positive (HPV⁺) cancers represent a distinct subset of HNSCC in terms of biology and clinical behaviour. In general, they are characterised by better overall survival and an improved response to conventional radio-chemotherapy (RT/CT) compared with HPV-negative (HPV⁻) cancers.^5,6 To further minimise treatment-related toxicity without compromising outcome, there have been suggestions of treatment de-escalation in conjunction with targeted therapies.⁷The novel anticancer agent TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) selectively kills several types of malignant cell lines with little effect on normal cells.⁸ Recombinant TRAIL or monoclonal antibodies targeting TRAIL receptors (TRAIL-Rs) are currently being tested in phase I/II clinical trials for patients with advanced tumours.^9,10 TRAIL induces cell death by binding to TRAIL-R1 or TRAIL-R2, resulting in receptor oligomerisation and formation of the death-inducing signalling complex (DISC)¹¹ and activation of initiator caspase-8.¹² Caspase-8 directly activates effector caspase-3 to induce apoptosis through the type I pathway or cleaves the BH3-only protein Bid, generating tBid. This type II pathway involves an amplification loop through the intrinsic pathway of apoptosis characterised by cytochrome c release from the mitochondria, activation of initiator caspase-9 and ultimately caspase-3.¹³Despite its tumour-selective activity, various cancer cell lines remain resistant to TRAIL, limiting the clinical potential of TRAIL-based monotherapies. Many recent studies focus on combination strategies with other agents to sensitise resistant cells to TRAIL.¹⁴ The proteasome inhibitor bortezomib is an FDA-approved drug for the treatment of multiple myeloma, but has shown only little single-agent activity in solid malignancies such as HNSCC while being effective in combination with other treatment options.^{15, 16, 17} Combining bortezomib with TRAIL-R agonists produced a synergistic cytotoxic effect in various types of cancers. Potential mechanisms underlying sensitisation to TRAIL-induced apoptosis include inhibition of NF-κB signalling, stabilisation of BH3-only proteins, p53 or p21, upregulation of TRAIL-Rs and enhanced stability of caspase-8.^{18, 19, 20, 21, 22, 23, 24, 25, 26}So far, little data is available on the therapeutic potential of TRAIL alone or in combination with bortezomib in HNSCC or other HPV⁺ related cancers. Treatment with the proteasome inhibitor MG132 sensitised TRAIL-resistant HPV⁺ cervical cancer cells to TRAIL through p53-dependent upregulation of TRAIL-Rs and inactivation of XIAP.²⁷ Overexpression of E6 was shown to protect colon cancer cells from death receptor-induced apoptosis by affecting the stability of the DISC, indicating a functional link between the presence of E6 and TRAIL signalling.²⁸In this study, we tested the response of HPV⁺ and HPV⁻ HNSCC cells to treatment with TRAIL alone or combined with bortezomib, revealing a clear pattern of sensitivity to TRAIL depending on HPV status and a synergistic effect when combined with bortezomib. In addition, we identified some of the proteins and pathways involved in the response to TRAIL/bortezomib in HNSCCs.     

Mitochondrial inhibitor sensitizes non-small-cell lung carcinoma cells to TRAIL-induced apoptosis by reactive oxygen species and Bcl-XL/p53-mediated amplification mechanisms

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy; however, non-small-cell lung carcinoma (NSCLC) cells are relatively TRAIL resistant. Identification of small molecules that can restore NSCLC susceptibility to TRAIL-induced apoptosis is meaningful. We found here that rotenone, as a mitochondrial respiration inhibitor, preferentially increased NSCLC cells sensitivity to TRAIL-mediated apoptosis at subtoxic concentrations, the mechanisms by which were accounted by the upregulation of death receptors and the downregulation of c-FLIP (cellular FLICE-like inhibitory protein). Further analysis revealed that death receptors expression by rotenone was regulated by p53, whereas c-FLIP downregulation was blocked by Bcl-X_L overexpression. Rotenone triggered the mitochondria-derived reactive oxygen species (ROS) generation, which subsequently led to Bcl-X_L downregulation and PUMA upregulation. As PUMA expression was regulated by p53, the PUMA, Bcl-X_L and p53 in rotenone-treated cells form a positive feedback amplification loop to increase the apoptosis sensitivity. Mitochondria-derived ROS, however, promote the formation of this amplification loop. Collectively, we concluded that ROS generation, Bcl-X_L and p53-mediated amplification mechanisms had an important role in the sensitization of NSCLC cells to TRAIL-mediated apoptosis by rotenone. The combined TRAIL and rotenone treatment may be appreciated as a useful approach for the therapy of NSCLC that warrants further investigation.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cancer therapeutic because it can selectively induce apoptosis in tumor cells in vitro, and most importantly, in vivo with little adverse effect on normal cells.¹ However, a number of cancer cells are resistant to TRAIL, especially highly malignant tumors such as lung cancer.^{2, 3} Lung cancer, especially the non-small-cell lung carcinoma (NSCLC) constitutes a heavy threat to human life. Presently, the morbidity and mortality of NSCLC has markedly increased in the past decade,⁴ which highlights the need for more effective treatment strategies.TRAIL has been shown to interact with five receptors, including the death receptors 4 and 5 (DR4 and DR5), the decoy receptors DcR1 and DcR2, and osteoprotegerin.⁵ Ligation of TRAIL to DR4 or DR5 allows for the recruitment of Fas-associated protein with death domain (FADD), which leads to the formation of death-inducing signaling complex (DISC) and the subsequent activation of caspase-8/10.⁶ The effector caspase-3 is activated by caspase-8, which cleaves numerous regulatory and structural proteins resulting in cell apoptosis. Caspase-8 can also cleave the Bcl-2 inhibitory BH3-domain protein (Bid), which engages the intrinsic apoptotic pathway by binding to Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist killer (BAK). The oligomerization between Bcl-2 and Bax promotes the release of cytochrome c from mitochondria to cytosol, and facilitates the formation of apoptosome and caspase-9 activation.⁷ Like caspase-8, caspase-9 can also activate caspase-3 and initiate cell apoptosis. Besides apoptosis-inducing molecules, several apoptosis-inhibitory proteins also exist and have function even when apoptosis program is initiated. For example, cellular FLICE-like inhibitory protein (c-FLIP) is able to suppress DISC formation and apoptosis induction by sequestering FADD.^{8, 9, 10, 11}Until now, the recognized causes of TRAIL resistance include differential expression of death receptors, constitutively active AKT and NF-κB,^{12, 13} overexpression of c-FLIP and IAPs, mutations in Bax and BAK gene.² Hence, resistance can be overcome by the use of sensitizing agents that modify the deregulated death receptor expression and/or apoptosis signaling pathways in cancer cells.⁵ Many sensitizing agents have been developed in a variety of tumor cell models.² Although the clinical effectiveness of these agents needs further investigation, treatment of TRAIL-resistant tumor cells with sensitizing agents, especially the compounds with low molecular weight, as well as prolonged plasma half-life represents a promising trend for cancer therapy.Mitochondria emerge as intriguing targets for cancer therapy. Metabolic changes affecting mitochondria function inside cancer cells endow these cells with distinctive properties and survival advantage worthy of drug targeting, mitochondria-targeting drugs offer substantial promise as clinical treatment with minimal side effects.^{14, 15, 16} Rotenone is a potent inhibitor of NADH oxidoreductase in complex I, which demonstrates anti-neoplastic activity on a variety of cancer cells.^{17, 18, 19, 20, 21} However, the neurotoxicity of rotenone limits its potential application in cancer therapy. To avoid it, rotenone was effectively used in combination with other chemotherapeutic drugs to kill cancerous cells.²²In our previous investigation, we found that rotenone was able to suppress membrane Na⁺,K⁺-ATPase activity and enhance ouabain-induced cancer cell death.²³ Given these facts, we wonder whether rotenone may also be used as a sensitizing agent that can restore the susceptibility of NSCLC cells toward TRAIL-induced apoptosis, and increase the antitumor efficacy of TRAIL on NSCLC. To test this hypothesis, we initiated this study.     

Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype

TNF-related apoptosis inducing ligand (TRAIL), a member of the TNF superfamily released by microglia, appears to be involved in the induction of apoptosis following focal brain ischemia. Indeed, brain ischemia is associated with progressive enlargement of damaged areas and prominent inflammation. As ischemic preconditioning reduces inflammatory response to brain ischemia and ameliorates brain damage, the purpose of the present study was to evaluate the role of TRAIL and its receptors in stroke and ischemic preconditioning and to propose, by modulating TRAIL pathway, a new therapeutic strategy in stroke. In order to achieve this aim a rat model of harmful focal ischemia, obtained by subjecting animals to 100 min of transient occlusion of middle cerebral artery followed by 24 h of reperfusion and a rat model of ischemic preconditioning in which the harmful ischemia was preceded by 30 mins of tMCAO, which represents the preconditioning protective stimulus, were used. Results show that the neuroprotection elicited by ischemic preconditioning occurs through both upregulation of TRAIL decoy receptors and downregulation of TRAIL itself and of its death receptors. As a counterproof, immunoneutralization of TRAIL in tMCAO animals resulted in significant restraint of tissue damage and in a marked functional recovery. Our data shed new light on the mechanisms that propagate ongoing neuronal damage after ischemia in the adult mammalian brain and provide new molecular targets for therapeutic intervention. Strategies aimed to repress the death-inducing ligands TRAIL, to antagonize the death receptors, or to activate the decoy receptors open new perspectives for the treatment of stroke.Stroke is a leading cause of death in industrialized countries¹ and the most frequent cause of disability in adults.² Although different mechanisms are involved in the pathogenesis of stroke, increasing evidence shows that ischemic injury and subsequent inflammation are responsible for damage progression,³ characterized by irreversible neuronal damage within minutes of the onset.In the past 30 years, it has been demonstrated that the brain''s resistance to ischemic injury can be transiently augmented by previous exposure to a non-injurious preconditioning (PC) stimulus.⁴ Evidence demonstrates that PC inhibits apoptosis in the penumbra region, thus preventing the spread of infarction. In addition, PC-induced neuroprotection appears related to a persistent activation of survival kinases in the penumbra.⁵ PC seems recognized by sensor molecules, such as neurotransmitters, cytokines, and others, as a sign of an event potentially more severe to come.⁶ In particular, inflammatory cytokines, beside representing PC sensor molecules, have a relevant role in acute stroke. For instance, tumor necrosis factor-α (TNF-α) and IL-1 mediate the inflammatory/immune response related to progression of the ischemic lesion.⁷ In the brain, cytokines are expressed not only in systemic immunocytes but also in resident cells, including neurons and glia.⁸ In particular, microglia have a key role as immune-competent cells of the CNS. Recruitment of leukocytes from the blood stream and activation of microglia are thought to contribute to the extension of the infarct core into the surrounding penumbra.⁵ Substantial evidence demonstrates that ischemia-activated microglia releases several pro-inflammatory cytokines, as well as other potentially cytotoxic molecules, including NO, ROS, and eicosanoids.⁹ Such a large arsenal of cytotoxic molecules appear to be involved in the induction of neuronal death.TNF-related apoptosis inducing ligand (TRAIL), a proapoptotic member of the TNF superfamily released by glia^{10, 11} and injured neurons,¹² appears to trigger apoptosis following focal brain ischemia.¹³ TRAIL binds five receptors, death receptor-4 (DR4), DR5, decoy receptor 1 (DcR1), DcR2, and osteoprogeterin. Although DR4 and DR5 receptors contain an intracellular death domain related to the apoptotic pathway, DcR1 and DcR2 serve as decoy receptors,¹⁴ eventually buffering death receptors'' binding of TRAIL, thus preventing apoptosis.^{15, 16}Although recent work attempted to establish a relationship between TRAIL pathway and brain ischemia,^{17, 18} only scant data are available on the role of TRAIL and its receptors in focal ischemia,¹⁹ and no data are known on the role of TRAIL in brain PC-induced neuroprotection.In the present study, we evaluated the role of TRAIL and its receptors in stroke and ischemic PC and verified the hypothesis of a potential mode for therapeutic intervention in stroke, by administrating a specific anti-TRAIL antibody in rats subjected to transient middle cerebral artery occlusion (tMCAO).     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.Hearing loss affects more than 360 million people worldwide and is often irreversible.¹ Mechanosensory hair cells (HCs), the receptor cells of hearing and balance, are not regenerated in the adult mammal and their death results in permanent hearing loss.^{2, 3} HCs are surrounded by glia-like supporting cells (SCs) that are necessary for HC survival and function (reviewed in Monzack et al.).⁴ SCs perform many functions, including providing critical trophic factors, preventing excitotoxicity, and mediating regeneration in those systems (non-mammalian vertebrates) capable of replacing lost HCs.^{5, 6, 7, 8, 9, 10, 11} When HCs die, SCs also preserve the integrity and function of the remaining tissue by forming scars and clearing dead HCs.^{2, 12, 13, 14, 15, 16, 17} Maintaining a fluid barrier at the surface of the sensory epithelium after damage is necessary to preserve the electro-chemical gradient that drives HC depolarization and therefore sensory transduction after the onset of hearing (reviewed in Wangemann).¹⁸Several major stressors cause HC death,^{19, 20, 21, 22} including aging, noise trauma, and exposure to therapeutic drugs with ototoxic side effects. When a HC is killed by noise or aminoglycoside antibiotics, surrounding SCs form a filamentous actin (F-actin) cable that constricts the HC at its apex.^{2, 12, 13, 14, 15, 16, 17} This process separates the apical portion of the cell, including the stereocilia bundle, from the HC body and preserves a sealed reticular lamina.²³ In the chick utricle, following the apical constriction of dead HCs, the SCs engulf and phagocytose the remaining HC corpse.¹⁵ Additional data from the chick indicate that the ototoxic drug cisplatin impairs some SC functions, including regeneration of HCs or clearance of HC debris.²⁴ We hypothesized that SCs would have significant phagocytic activity in the mature mammalian inner ear, and that cisplatin would impair this activity. To examine these dynamic processes, we live-imaged SC phagocytic activity in the adult mouse utricle and compared the SC responses with HC stress and death caused by aminoglycosides versus cisplatin.     

Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.Apart from the apoptotic cell death pathway that ligands of the tumor necrosis factor (TNF) family can activate, these ligands and various other inducers, including the interferons and various pathogen components, have in recent years been found also to trigger a signaling cascade that induces programmed necrotic death (necroptosis). This cascade encompasses sequential activation of the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL).^{1, 2, 3, 4, 5} RIPK3-mediated phosphorylation of MLKL triggers its oligomerization, which is necessary and sufficient for the induction of cell death,^{6, 7, 8} and can also trigger some non-deadly functions.⁹ MLKL was recently suggested to trigger cell death by binding to cellular membranes and initiating ion fluxes through them.^{6, 7, 8, 10} However, its exact molecular target in death induction is contentious.^{6, 8, 10, 11, 12} Current knowledge of the subcellular sites of MLKL action is based mainly on determination of the location of this protein close to the time of cell death. Here we present a detailed assessment of the cellular location of MLKL at different times following its activation. Our findings indicate that before cell death, MLKL translocates to the nucleus along with RIPK1 and RIPK3.     

p62/SQSTM1 upregulation constitutes a survival mechanism that occurs during granulocytic differentiation of acute myeloid leukemia cells

The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34⁺ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.^{1, 2} Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.^{3, 4, 5, 6, 7} p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.^{8, 9} At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).^{3, 5, 6, 7, 10, 11, 12, 13} p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL¹⁴ and Wnt pathways^{15, 16} or by inducing the activation of caspase 8, a pro-death protein.^{17, 18} Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.¹⁰ In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.¹⁹ In contrast, p62/SQSTM1 favors differentiation of osteoclasts,²⁰ osteoblasts,²¹ neurons,²² megakaryocytes²³ and macrophages.²⁴ The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.^{25, 26} Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.^{27, 28} All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.^{29, 30, 31} Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.^{32, 33, 34}Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.³⁵ In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes.