Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.     

Modulation of A1 and A2B adenosine receptor activity: a new strategy to sensitise glioblastoma stem cells to chemotherapy

Therapies that target the signal transduction and biological characteristics of cancer stem cells (CSCs) are innovative strategies that are used in combination with conventional chemotherapy and radiotherapy to effectively reduce the recurrence and significantly improve the treatment of glioblastoma multiforme (GBM). The two main strategies that are currently being exploited to eradicate CSCs are (a) chemotherapeutic regimens that specifically drive CSCs toward cell death and (b) those that promote the differentiation of CSCs, thereby depleting the tumour reservoir. Extracellular purines, particularly adenosine triphosphate, have been implicated in the regulation of CSC formation, but currently, no data on the role of adenosine and its receptors in the biological processes of CSCs are available. In this study, we investigated the role of adenosine receptor (AR) subtypes in the survival and differentiation of CSCs isolated from human GBM cells. Stimulation of A₁AR and A_2BAR had a prominent anti-proliferative/pro-apoptotic effect on the CSCs. Notably, an A₁AR agonist also promoted the differentiation of CSCs toward a glial phenotype. The differential effects of the two AR agonists on the survival and/or differentiation of CSCs may be ascribed to their distinct regulation of the kinetics of ERK/AKT phosphorylation and the expression of hypoxia-inducible factors. Most importantly, the AR agonists sensitised CSCs to the genotoxic activity of temozolomide (TMZ) and prolonged its effects, most likely through different mechanisms, are as follows: (i) by A_2BAR potentiating the pro-apoptotic effects of TMZ and (ii) by A₁AR driving cells toward a differentiated phenotype that is more sensitive to TMZ. Taken together, the results of this study suggested that the purinergic system is a novel target for a stem cell-oriented therapy that could reduce the recurrence of GBM and improve the survival rate of GBM patients.Glioblastoma multiforme (GBM), classified as grade IV on the World Health Organization scale,¹ is the most common type of primary malignant brain tumour.² The current therapeutic strategy includes surgery followed by radiation and chemotherapy using temozolomide (TMZ). This therapeutic approach slightly improves the survival rate of GBM patients, but their prognosis remains poor and most patients die of tumour recurrence.³ The causes of the recurrence of GBM are complex and include the high proliferative index of the tumour cells and their resistance to chemotherapy and radiotherapy, particularly in the case of the cancer stem cells (CSCs). These cells have been proposed to not only initiate the genesis of GBM and contribute to its highly proliferative nature, but to also be the basis for its recurrences following treatment. Moreover, it has been reported that the most aggressive or refractory cancers contain the highest number of CSCs.^{4, 5, 6}These findings suggest that innovative stem cell-orientated therapy may be an effective strategy to reduce tumour recurrence and significantly improve GBM treatment outcomes.^{7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18} This type of therapy may not be easy to implement because CSCs have been shown to have a low level of reactive oxygen species¹⁹ and to be more resistant to ionising radiation,²⁰ vincristine,²¹ hypoxia and other chemotherapeutics²² compared with non-CSCs. In contrast, the preferential elimination of the CSC population may contribute to the effectiveness of TMZ, which is the most effective pharmacologic agent used in glioma treatment;²³ however, the activity of TMZ appears to be short lived because the drug causes the reversible blockage of the cell cycle of CSCs.²⁴ Moreover, long-term TMZ therapy results in the occurrence of drug-resistant GBM cells,²⁵ indicating the need to develop distinct strategies to overcome this resistance.Extracellular purines have been implicated in several aspects of GBM biology, such as proliferation,²⁶ migration,²⁷ invasion²⁸ and death.²⁹ The concentration of adenosine in the extracellular fluid of glioma tissue was reported to be in the low micromolar range,³⁰ which is sufficiently high to stimulate all the four of the adenosine receptor (AR) subtypes (A₁, A_2A, A_2B and A₃).³¹ Each of the ARs have a pivotal role in the control of tumour growth and invasiveness^{32, 33, 34} but to date, no data on their role in CSC biology are available. Recently, it was demonstrated that treatment with adenosine triphosphate reduced the rate of sphere formation by glioma cells and that purinergic receptors are differentially expressed in spheres of tumour cells and adherent cells.³³ In this study, we investigated the role of AR subtypes in the survival and differentiation of CSCs. Globally, our data clarified the role of each AR subtype in CSC functionality and suggested that the purinergic system is a novel pharmacological target for the development of new anti-CSC therapies, particularly those aimed at the treatment of GBM recurrences.     

β-Catenin and NF-κB co-activation triggered by TLR3 stimulation facilitates stem cell-like phenotypes in breast cancer

Cancer stem cells (CSCs) are responsible for tumor initiation and progression. Toll-like receptors (TLRs) are highly expressed in cancer cells and associated with poor prognosis. However, a linkage between CSCs and TLRs is unclear, and potential intervention strategies to prevent TLR stimulation-induced CSC formation and underlying mechanisms are lacking. Here, we demonstrate that stimulation of toll-like receptor 3 (TLR3) promotes breast cancer cells toward a CSC phenotype in vitro and in vivo. Importantly, conventional NF-κB signaling pathway is not exclusively responsible for TLR3 activation-enriched CSCs. Intriguingly, simultaneous activation of both β-catenin and NF-κB signaling pathways, but neither alone, is required for the enhanced CSC phenotypes. We have further identified a small molecule cardamonin that can concurrently inhibit β-catenin and NF-κB signals. Cardamonin is capable of effectively abolishing TLR3 activation-enhanced CSC phenotypes in vitro and successfully controlling TLR3 stimulation-induced tumor growth in human breast cancer xenografts. These findings may provide a foundation for developing new strategies to prevent the induction of CSCs during cancer therapies.Despite incessant efforts to combat cancer over decades, breast cancer is still the second leading cause of death in women, remaining high with over 39 000 deaths in 2012 in the United States alone.¹ Conventional interventions, such as radiation or chemotherapy, may eliminate the bulk of the tumor but spare rare aggressive cancer cells that have an exceptional capacity to survive, self-renew, and advance the malignancy. These residual tumor cells have recently been found to possess key stem-like properties and have thus been termed ‘cancer stem cells (CSCs)''.^{2, 3, 4, 5} Breast CSCs, characterized by expression of CD44^high/CD24^−/low surface markers, are proposed to be largely responsible for cancer progression and metastasis.^3,6,7 These CD44^high/CD24^−/low cells possess stem cell-like properties and tumor-initiating capacity. Furthermore, these cells resist standard therapies^3,6,8,9 and can be converted from non-CSC cells under certain conditions.^10,11 Therefore, specific targeting of CSCs within a tumor will be imperative to prevent disease progression and recurrence.⁵ However, the conditions and mechanisms underlying CSC formation remain poorly understood. Although the majority of cancers arise from de novo oncogenic and epigenetic alterations, most tumors display signals of unremitting inflammatory activity,¹² which occurs even in the absence of infection or autoimmunity.¹³Toll-like receptors (TLRs) are a key family of microbial sensors in the host innate and adaptive immunity as well as in tissue repair and regeneration. They are also involved in the inflammatory signaling triggered by endogenous macromolecules released by injured tissue.^14,15 Ten TLRs are encoded by the human genome. TLRs detecting nucleic acids (TLR3, TLR7, TLR8, and TLR9) are localized in the endosomal compartment in nearly all cell types, while TLRs mainly detecting proteolipidic structures (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) are exposed on the cell surface.^14,16 In cancer, TLRs have emerged as important participants in tumorigenesis. TLR3, 4, 7, and 9 were overexpressed in 70, 72, 67, and 78% of patients with esophageal cancer.¹⁷ The -196 to -174del/del genotype of TLR2 may increase the risk of gastric cancer,¹⁸ and TLR4+896A>G polymorphism is a risk factor for non-cardia gastric carcinoma.¹⁹ Functions of epithelial-expressed TLR2 and 5 in promoting epithelial cell survival, proliferation, migration,²⁰ and angiogenesis (TLR2 only)²¹ may be usurped by tumor cells to facilitate progression and metastasis. Although TLR3, 5, 7, 8, and 9 may achieve antitumor effects by converting immune tolerance into antitumor immunity,¹⁴ considerable discrepancies have been reported. For instance, high TLR3 expression in esophageal cancer cells was significantly associated with a higher probability of lymph-node metastasis and increased depth of invasion.¹⁷ Elevated TLR3 expression in breast cancer was also associated with poor prognosis.^22,23Several clinical trials using TLR agonists for cancer treatment are currently in progress. Among all anticancer immunotherapy agents, TLR agonists are classified as the ones with highest potential. However, clinical outcomes are inconsistent and repeatedly disappointing.²⁴ Specifically, high expectations were placed on TLR3 agonists for their ability to boost host immune systems to fight diseases. TLR3 is located in intracellular endosomes for the recognition of double-stranded RNA (dsRNA) and polyinosinic-polycytidylic acid (poly(I:C), a synthetic analog of dsRNA).²⁵ In addition to upregulating immune response, a broader range of functions of TLR3 have been revealed recently, especially in stem cells. For instances, activation of TLR3 was found to amplify mesenchymal stem cell trophic factors and enhance therapeutic potency.²⁶ Recently, Lee et al.²⁷ also showed that TLR3 stimulation caused rapid and global changes in the expression of epigenetic modifiers to enhance chromatin remodeling and nuclear reprogramming when converting adult cells to induced pluripotent stem cells. Nevertheless, the role of TLR3 in cancer remains inconsistent, and its function in breast CSCs is unclear.Here, we demonstrate that TLR3 activation in breast cancer cells leads to a preferential enrichment of a subset of cells with CSC phenotypes in vitro and in vivo. Conventional NF-κB signaling is not fully responsible for the enhanced CSC properties. Unexpectedly, β-catenin pathway is required for the promotion of CSC phenotypes in breast cancer cells following TLR3 activation. Our results provide new tantalizing strategies to effective target breast and other CSCs with elevated TLR3 expression to prevent progression and relapse.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Targeting proapoptotic protein BAD inhibits survival and self-renewal of cancer stem cells

Emerging evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies is one of the major limiting factors of cancer therapy efficacy. Identification of mechanisms responsible for survival and self-renewal of CSC will help design new therapeutic strategies that target and eliminate both differentiated cancer cells and CSC. Here we demonstrated the potential role of proapoptotic protein BAD in the biology of CSC in melanoma, prostate and breast cancers. We enriched CD44⁺/CD24⁻ cells (CSC) by tumorosphere formation and purified this population by FACS. Both spheres and CSC exhibited increased potential for proliferation, migration, invasion, sphere formation, anchorage-independent growth, as well as upregulation of several stem cell-associated markers. We showed that the phosphorylation of BAD is essential for the survival of CSC. Conversely, ectopic expression of a phosphorylation-deficient mutant BAD induced apoptosis in CSC. This effect was enhanced by treatment with a BH3-mimetic, ABT-737. Both pharmacological agents that inhibit survival kinases and growth factors that are involved in drug resistance delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. Furthermore, the frequency and self-renewal capacity of CSC was significantly reduced by knocking down the BAD expression. Consistent with our in vitro results, significant phosphorylation of BAD was found in CD44⁺ CSC of 83% breast tumor specimens. In addition, we also identified a positive correlation between BAD expression and disease stage in prostate cancer, suggesting a role of BAD in tumor advancement. Our studies unveil the role of BAD in the survival and self-renewal of CSC and propose BAD not only as an attractive target for cancer therapy but also as a marker of tumor progression.Although tumors initially respond positively to anti-cancer agents, several cancers, despite the best care and significant improvements in treatment, recur and progress to advanced stages of the disease. The mechanisms underlying this recurrence and metastasis are not clearly understood. Over the past decade, substantial evidence supported the cancer stem cell (CSC) hypothesis as a viable explanation for the initiation, progression and recurrence of cancer. According to this hypothesis, each tumor harbors a small subpopulation of specialized cells among cellular heterogeneity, known as CSC. These cells exhibit self-renewal property that drives tumorigenesis and plasticity to differentiate into multiple cell types contributing to tumor cellular heterogeneity. Support for this hypothesis came from the studies by Lapidot et al. who identified tumor-initiating cells in acute myeloid leukemia.^{1, 2} Subsequently, CSCs have been identified in several cancers.^{3, 4, 5, 6, 7, 8, 9, 10}Accumulating evidence suggests that current cancer therapies can only shrink tumors as they target and kill the differentiated cancer (DC) cells, but are unable to target the rare CSC population.^{11, 12} Thus, despite a wealth of information on DC cells, the active survival and self-renewal pathways in CSCs have not been characterized thoroughly. An understanding of the molecular mechanisms involved in the survival, self-renewal and resistance of CSCs to current therapeutic regimens is of immense clinical interest. This information will help in developing novel strategies for more effective treatments for cancer.Most anti-cancer drugs exert their effects through triggering the apoptotic pathways. However, malignant cancer cells can escape apoptosis by altering the expression level of proapoptotic and antiapoptotic BCL-2 family members. Considering the potential role of BCL-2 family members in tumorigenesis and cancer cell survival, their role in CSC biology has been increasingly studied.^{13, 14} BAD (BCL2-antagonist of cell death) is a member of the BH3-only BCL-2 family protein that when dephosphorylated promotes apoptosis by heterodimerizing with the antiapoptotic proteins BCL-XL and BCL-2.¹⁵ The cytotoxic effects of BAD are controlled by mechanisms that regulate its phosphorylation on at least two distinct serine residues, S112 and S136.^{16, 17, 18} Previously, we showed that phosphorylation at either site is sufficient to protect prostate cancer cells from apoptosis.^{19, 20, 21} We also showed that BAD promotes prostate tumor growth in mouse models.²² Clinically, while BAD expression was associated with relapse in tamoxifen-treated breast cancer patients,^{23, 24} phospho-BAD expression was associated with cisplatin resistance and poor overall survival in ovarian cancer.²⁵Our previous findings along with other reports showing the role of BAD in the apoptosis modulation and growth of DC cells^{19, 22, 26} prompted us to explore the potential role of BAD in the biology of CSCs. We started our investigation by assessing the role of BAD in survival and self-renewal of CSCs. As we observed a significant role for BAD in CSC''s biology, we extended our work to assess the BAD phosphorylation in CSCs of breast cancer patient tumors and for a potential correlation between BAD expression and disease progression in prostate cancer.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

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.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Enrichment of c-Met+ tumorigenic stromal cells of giant cell tumor of bone and targeting by cabozantinib

Giant cell tumor of bone (GCTB) is a very rare tumor entity, which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis, emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells, stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation, differentiation, migration, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, immunohistochemistry, antibody protein array, Alu in situ hybridization, FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies, differentiated to osteoblasts, migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4, indicating self-renewal, invasion and differentiation potential. Compared with adherent-growing cells, markers for pluripotency, stemness and cancer progression, including the CSC surface marker c-Met, were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib, an inhibitor of c-Met in phase II trials, eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met⁺ tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB.Giant cell tumor of bone (GCTB) is a very rare, osteolytic neoplasm deemed histologically benign, but it is locally aggressive and destroys bone and overlying soft tissue.^1,2 Surgery has been the preferred treatment for GCTB; however, the lesion tends to recur locally. In ~6% of cases, the development of lung metastases has been observed.^{3, 4, 5} GCTB has a predilection for the epiphyseal/metaphyseal region of long bones and the spine and thus can cause substantial morbidity.⁶ For patients with unresectable GCTB, the use of chemotherapeutics, bisphosphonates, radiation, radiofrequency thermal ablation and arterial embolization are palliative options with limited effects on tumor control.^{7, 8, 9} Recently, denosumab, a RANKL inhibitor, has been approved for GCTB, and it targets, especially the neoplastic stromal cells, which express high concentrations of RANKL.^9,10GCTB is composed of three different cell types: multinucleated, osteoclast-like giant cells, CD68⁺ phagocytic histiocytes and fibroblast-like stromal cells. The stromal cells have been identified as the neoplastic cell population,^{11, 12, 13} and it is believed that they develop from mesenchymal stem cells (MSCs).^14,15 The latter notion is supported by studies that demonstrate involvement of MSCs in tumor development—for example, in the development of sarcoma.¹⁶According to the hypothesis, cancer stem cells (CSCs) are responsible for growth, invasion, metastasis and therapy resistance of cancer, because this small sub-population within the tumor mass is thought to survive conventional cytotoxic therapy because of activated defense and survival mechanisms.¹⁷ CSCs are characterized by self-renewal potential and the ability to differentiate, thereby generating a heterogeneous cell population of the originating tumor.^{18, 19, 20} In addition, CSCs are proposed to mediate uncontrolled growth, therapy resistance, invasion and metastasis.²¹ Markers for CSCs have been identified in various tumor entities, and the selected marker-positive fractions can reconstitute the original tumor in immunodeficient mice.²² There are several surface markers for CSCs of different tumor entities and the c-Met marker represents such a typical CSC sub-population.^{23, 24, 25}c-Met belongs to the group of receptor tyrosine kinases and has a key role in cell survival, growth, angiogenesis and metastasis.²⁶ c-Met and its physiologic ligand hepatocyte growth factor (HGF) are required for normal mammalian development and have an important role in epithelial–mesenchymal interactions during organ morphogenesis.²⁶ The intracellular signaling cascades activated by c-Met include the RAS-MAPK and PI3K-AKT pathways, as well as NF-κB and Wnt/GSK-3β/β-catenin signaling.²⁶ Many carcinomas overexpress c-Met, and the surrounding stroma overexpresses HGF. Currently, the therapeutic potential of the c-Met inhibitor cabozantinib (XL184) is intensively investigated. Cabozantinib is a potent dual inhibitor of c-Met and VEGFR-2 signaling.^25,27 The clinical efficacy of cabozantinib in several progressed tumor entities is under investigation in randomized phase II studies.²⁸ At the end of 2012, cabozantinib (Cometriq) was approved by the FDA for the treatment of patients with progressive medullary thyroid carcinoma.²⁹ Cabozantinib shows promise in preventing prostate cancer spread to bone because tumors were reduced on bone scans, and bone pain decreased after patients received cabozantinib.³⁰ These data may be of importance for GCTB, but until now, cabozantinib has not been investigated for the treatment of primary bone tumors.In the present study, we demonstrate that a c-Met⁺ sub-population of low-passage stromal cells isolated from eight freshly resected GCTB specimens possess self-renewal, differentiation and migratory potential, as well as the ability to form tumors in vivo. By comparing attached-growing c-Met^low and spheroidal c-Met^high cultures, we identified enhanced pluripotency, stemness and progression, as well as the enrichment of a c-Met⁺ population. Most importantly, cabozantinib strongly inhibited the self-renewal potential and in vivo growth of GCTB stromal cells. Thus, cabozantinib may be considered an effective future therapeutic option for the targeted elimination of a tumorigenic stromal sub-population in non-resectable or recurrent GCTB.     

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF)

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Osteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis through blood.¹ Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%.^{2, 3} However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs.^{4, 5} Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma.^{6, 7} Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.^{8, 9}In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation.¹⁰ Increasing evidence suggests that inflammation is closely related to tumorigenesis.¹¹ MIF plays a bridging role between inflammation and tumorigenesis.^{12, 13, 14} MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis.¹⁵ Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis.^{16, 17, 18} Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis.^{19, 20, 21} MIF has been well established to be involved in the development of glioblastoma,²² breast cancer,²³ bladder cancer²⁴ and colon cancer.^{20, 25} MIF was also upregulated in osteosarcoma.^{26, 27} The knockdown of MIF blocked osteosarcoma cell proliferation and invasion.²⁶ However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Wu et al. ²³ have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma.⁶ These controversial results prompted us to confirm the role of MIF in drug resistance in osteosarcoma.In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced reactive oxygen species (ROS)-mediated autophagy to rescue cell death. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.     

Tyr1068-phosphorylated epidermal growth factor receptor (EGFR) predicts cancer stem cell targeting by erlotinib in preclinical models of wild-type EGFR lung cancer

Tyrosine kinase inhibitors (TKIs) have shown strong activity against non-small-cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations. However, a fraction of EGFR wild-type (WT) patients may have an improvement in terms of response rate and progression-free survival when treated with erlotinib, suggesting that factors other than EGFR mutation may lead to TKI sensitivity. However, at present, no sufficiently robust clinical or biological parameters have been defined to identify WT-EGFR patients with greater chances of response. Therapeutics validation has necessarily to focus on lung cancer stem cells (LCSCs) as they are more difficult to eradicate and represent the tumor-maintaining cell population. Here, we investigated erlotinib response of lung CSCs with WT-EGFR and identified EGFR phosphorylation at tyrosine1068 (EGFR^tyr1068) as a powerful biomarker associated with erlotinib sensitivity both in vitro and in preclinical CSC-generated xenografts. In contrast to the preferential cytotoxicity of chemotherapy against the more differentiated cells, in EGFR^tyr1068 cells, erlotinib was even more active against the LCSCs compared with their differentiated counterpart, acquiring potential value as CSC-directed therapeutics in the context of WT-EGFR lung cancer. Although tumor growth was inhibited to a similar extent during erlotinib or chemotherapy administration to responsive tumors, erlotinib proved superior to chemotherapy in terms of higher tolerability and reduced tumor aggressiveness after treatment suspension, substantiating the possibility of preferential LCSC targeting, both in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) tumors. We conclude that EGFR^tyr1068 may represent a potential candidate biomarker predicting erlotinib response at CSC-level in EGFR-WT lung cancer patients. Finally, besides its invariable association with erlotinib sensitivity in EGFR-WT lung CSCs, EGFR^tyr1068 was associated with EGFR-sensitizing mutations in cell lines and patient tumors, with relevant diagnostic, clinical and therapeutic implications.Non-small-cell lung cancer (NSCLC) accounts for ∼80% of lung cancer subtypes and is the leading cause of cancer-related death worldwide.¹ In recent years, molecular characterization of NSCLC has reached an unprecedented detail and has allowed segregating NSCLC into discrete molecular subgroups, characterized by specific oncogenic drivers, such as epidermal growth factor receptor (EGFR), BRAF, KRAS, epidermal growth factor receptor 2 (HER2) mutations, MET amplification and anaplastic lymphoma kinase gene rearrangements (ALK).^{2, 3} Consequently, the understanding of NSCLC biology has brought two new classes of targeted agents into the clinical setting: EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors.^{4, 5} In particular, clinical trials have shown that NSCLC patients whose tumors harbor sensitizing EGFR mutations significantly benefit from the upfront use of an EGFR TKI, rather than conventional chemotherapy.^{6, 7, 8, 9, 10, 11} Although licensed for clinical use in chemotherapy-pretreated patients, regardless of EGFR mutational status, the EGFR TKI erlotinib has limited efficacy when compared with standard chemotherapy in patients with WT-EGFR NSCLC.^{12, 13, 14}However, a fraction of patients on erlotinib treatment may achieve clinically significant objective responses and prolonged disease control, despite the lack of detectable EGFR mutations.¹⁵ Nevertheless, no biomarker investigated so far was felt sufficiently robust to select for the use of erlotinib in the maintenance or refractory setting.¹⁶ Thus, it would be crucial to identify molecular predictors of TKI sensitivity in EGFR wild-type (WT) tumors in order to prospectively select the subgroup of patients who may benefit from erlotinib therapy. Moreover, EGFR TKIs have also shown a modest therapeutic effect in lung squamous cell carcinoma (SCC), where EGFR mutations are very rare and patients have limited therapeutic options in the maintenance and relapsed settings.^{16, 17, 18, 19, 20}Even more importantly, in order to obtain meaningful clinical responses it is crucial to effectively target the population of cells that are able to escape treatment and maintain the growth of a resistant tumor.²¹ Cancer stem cells (CSCs) have been in fact identified within most solid tumors, including lung tumors, and are associated with increased resistance to therapies.^{22, 23, 24, 25, 26, 27, 28, 29, 30} Thus, the efficacy of innovative therapeutic strategies should be validated against these more aggressive, tumor-maintaining cells.^{23, 27, 31} Importantly, TKI response has never been determined at the level of the tumor-maintaining CSCs. Thus, we investigated erlotinib response of EGFR mutation-negative lung cancer stem cells (LCSCs) and LCSC-based xenografts with the attempt to evaluate their sensitivity to the drug and correlate it with their molecular pattern in order to identify potential biomarkers predictive of erlotinib response in a WT-EGFR context at the CSC level.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.