The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.Apoptosis is an indispensible process to maintain cellular homeostasis, in particular in highly dynamic tissues. Apoptosis can be induced by activation of death receptors (DRs; such as TRAIL-R1/R2 or cluster of differentiation 95 (CD95)) or by intrinsic disturbance of mitochondria.¹ Death ligands (DLs; TNF-related apoptosis-inducing ligand (TRAIL) or CD95L), when bound to their respective DRs, induce apoptosis by activation of procaspase-8 within the death-inducing signalling complex (DISC).² Caspase-8 activation is followed by proteolytic cleavage of caspase-3.³ Extrinsic and intrinsic cell death is negatively controlled by caspase inhibitors such as X-linked inhibitor of apoptosis protein (XIAP)⁴ or by B-cell lymphoma 2 (Bcl-2) proteins that suppress the mitochondria outer membrane permeability (MOMP) by limiting Bax (Bcl-2-associated X protein)/Bak (Bcl-2 homologous antagonist/killer) translocation into the mitochondrial outer membrane.⁵ The extrinsic signalling cascade communicates with the intrinsic death pathway by cleavage of Bid (BH3 interacting-domain death agonist), a pro-apoptotic member of the BH3 (Bcl-2 homology domain 3)-only subfamily of Bcl-2 proteins.¹ Other stimuli such as genotoxic stress allow for translocation and pore formation of pro-apoptotic multidomain Bcl-2 proteins Bax and Bak in the outer mitochondrial membrane.^{6, 7, 8} This process promotes release of mitochondria-derived apoptogenic proteins, in particular cytochrome c,⁹ or smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI).¹⁰ Within the apoptosome,¹¹ active caspase-9 finally leads to activation of caspase-3,¹² and subsequent cell death.Anti-apoptotic multidomain Bcl-2 proteins (Bcl-2, Bcl-2-like protein 2 (Bcl-w), B-cell lymphoma-extra large (Bcl-X_L), induced myeloid leukaemia cell differentiation protein (Mcl-1) and Bcl-2-related protein A1 (A1)) with four Bcl-2 homology domains (BH1, BH2, BH3 and BH4) suppress the pro-apoptotic function of Bax-like proteins such as Bax, Bak and Bok (that contain BH1–BH3 domains) or the BH3-only proteins Bad (Bcl-2-associated death promoter), Bim (Bcl-2-like protein 11), Bid, Noxa (phorbol-12-myristate-13-acetate-induced protein 1) and Puma (p53 upregulated modulator of apoptosis).¹³ Regulation of mitochondria-mediated apoptosis is determined by the balance between pro- and anti-apoptotic Bcl-2 proteins.¹⁴In a variety of cancer types, a decrease of BH3-only protein or upregulation of pro-survival Bcl-2 proteins is associated with poor prognosis.¹⁵ In metastatic squamous cell carcinoma (SCC) of the skin or the so-called ‘head and neck SCC'' (HNSCC), high expression of pro-survival Bcl-2 proteins conferred radio- and chemotherapy resistance.^{16, 17} These findings mark Bcl-2 proteins as regulators of SCC apoptosis and indicate that BH3 mimetics may hold therapeutic potential for metastatic SCC. The BH3 mimetics navitoclax (ABT263) and ABT199 are currently under investigation in clinical studies.^{18, 19, 20} Mechanistically, their lead compound ABT737 suppresses Bcl-2 activity by binding to the hydrophobic groove of Bcl-2, Bcl-w and Bcl-X_L.¹⁸ As ABT263 upregulates Mcl-1, resistance to a number of Bcl-2 inhibitors (ABT737 and ABT263) has been described.²¹ Another compound, Obatoclax, was developed to block all anti-apoptotic Bcl-2 proteins including Mcl-1.²² Obatoclax blocks the interaction of Bim or Bax with Mcl-1.²³ In this report, we have studied the effect of ABT737 for cell death in SCC of the skin and investigated the molecular mechanisms of resistance to different BH3 mimetics.     

Role of Ku70 in deubiquitination of Mcl-1 and suppression of apoptosis

Mcl-1 is a unique antiapoptotic Bcl2 family member with a short half-life due to its rapid turnover through ubiquitination. We discovered that Ku70, a DNA double-strand break repair protein, functions as a deubiquitinase to stabilize Mcl-1. Ku70 knockout in mouse embryonic fibroblast (MEF) cells or depletion from human lung cancer H1299 cells leads to the accumulation of polyubiquitinated Mcl-1 and a reduction in its half-life and protein expression. Conversely, expression of exogenous Ku70 in Ku70^−/− MEF cells restores Mcl-1 expression. Subcellular fractionation indicates that Ku70 extensively colocalizes with Mcl-1 in mitochondria, endoplasmic reticulum and nucleus in H1299 cells. Ku70 directly interacts with Mcl-1 via its C terminus (that is, aa 536–609), which is required and sufficient for deubiquitination and stabilization of Mcl-1, leading to suppression of apoptosis. Purified Ku70 protein directly deubiquitinates Mcl-1 by removing K48-linked polyubiquitin chains. Ku70 knockdown not only promotes Mcl-1 turnover but also enhances antitumor efficacy of the BH3-mimetic ABT-737 in human lung cancer xenografts. These findings identify Ku70 as a novel Mcl-1 deubiquitinase that could be a potential target for cancer therapy by manipulating Mcl-1 deubiquitination.Mcl-1 is an antiapoptotic molecule that is overexpressed in various types of cancers, including lung cancer,¹ leukemia,² lymphoma,³ hepatocellular carcinoma⁴ and so on. In addition to its antiapoptotic function, Mcl-1 is also an oncoprotein that promotes the development of cancer.⁵ In contrast to other Bcl2 family members such as Bcl2 and Bcl-XL, Mcl-1 is unique in its short half-life (30 min–3 h) and short-term prosurvival function, which probably relates to the presence of a long proline-, glutamic acid-, serine- and threonine-rich (PEST) region upstream of the Bcl2 homology (BH) domain.¹ The mechanism(s) that stabilizes the Mcl-1 protein are critical for its long-term survival function. Mcl-1 protein can be phosphorylated at multiple sites that distinctly regulate Mcl-1 protein turnover. For example, extracellular signal-regulated kinase 1/2-mediated T163 site phosphorylation enhances the half-life and antiapoptotic function of Mcl-1.^{1, 6} In contrast, S159 phosphorylation by GSK-3β facilitates Mcl-1 ubiquitination and degradation to reduce its survival activity.⁷Ubiquitination and deubiquitination are two reversible processes that can control protein stability. E3 ligases and deubiquitinases (deubiquitinating enzymes (DUBs)) are two groups of regulatory enzymes that orchestrate the ubiquitination levels of target proteins in eukaryotic cells.⁸ Recently, Mule and FBW7 have been identified as Mcl-1 ubiquitin E3 ligases that can directly induce polyubiquitination and degradation of Mcl-1.^{9, 10} Inversely, USP9X has been demonstrated as the Mcl-1 deubiquitinase that removes the Lys 48-linked polyubiquitin chains that normally mark Mcl-1 for proteasomal degradation, leading to stabilization of Mcl-1.³ Therefore, the stability of Mcl-1 in cells is tightly regulated by its E3 ligases and deubiquitinase, which is dependent on Mcl-1 phosphorylation status.^{3, 11}Ku70 is a protein that binds to DNA double-strand break (DSB) ends and is required for the non-homologous end-joining pathway of DSB repair.^{12, 13, 14, 15} The Ku70 protein consists of three structural domains, including the N-terminal, central (that is, DNA binding) and C-terminal domains.^{16, 17} Ku70 usually heterodimerizes with Ku86, which forms a functional complex for DSB repair. By forming a bridge between the broken DNA ends, the Ku70/Ku86 heterodimer acts to structurally support and align the DNA ends, to protect them from degradation and to prevent promiscuous binding to unbroken DNA. Ku70/Ku86 effectively aligns the DNA, while still allowing access of polymerases, nucleases and ligases to the broken DNA ends to promote end joining.¹⁸ In some cases, a fourth domain is present at the C terminus of Ku86, which binds to the DNA-dependent protein kinase catalytic subunit.¹⁹ Importantly, Ku70 also regulates apoptosis independent of its DSB repair activity. For example, a recent report revealed that Ku70 regulates the proapoptotic function of Bax by sequestering Bax from the mitochondria and mediating Bax deubiquitylation.²⁰ Here we discovered that Ku70 functions as a novel Mcl-1 deubiquitinase that directly removes polyubiquitin chains from Mcl-1 protein, leading to reduced Mcl-1 ubiquitination/degradation, enhanced stability and suppression of apoptosis.     

Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca²⁺ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca²⁺-dependent ROS production.The Bcl-2 proteins are a family of molecules comprised of both pro- and antiapoptotic members essential for the regulation of apoptotic cell death. In the classical paradigm, the antiapoptotic proteins Bcl-2, Bcl-x_L and Mcl-1, inhibit cell death during receipt of apoptotic stimuli by binding and sequestering the proapoptotic members.¹ It is now appreciated, however, that in the absence of apoptotic stimuli, Bcl-2 proteins have numerous non-canonical interactions that influence diverse cellular functions, although the precise mechanisms are poorly understood.² Since antiapoptotic Bcl-2 family members are frequently upregulated in cancer, determining if and how these non-canonical interactions confer survival or other advantages to the cancer cell, will be an important step toward identifying new therapeutic targets. One such interaction is with the outer mitochondrial membrane-localized voltage-dependent anion channel (VDAC), a porin channel with three isoforms that serves as a major diffusion pathway for ions and metabolites,³ and whose gating properties are affected by either Bcl-2 or Bcl-x_L binding.^{4, 5, 6}We recently identified an important role for Bcl-x_L/VDAC interactions in the regulation of mitochondrial [Ca²⁺].⁷ Moving Ca²⁺ from the cytoplasm to the mitochondrial matrix requires transfer across the outer membrane by VDAC^3,8 and across the inner membrane by the Ca²⁺ uniporter.⁹ Our studies showed that Bcl-x_L interacts with VDAC to facilitate Ca²⁺ uptake into the mitochondrial matrix. It is not known if other Bcl-2 family members, particularly Bcl-2 and Mcl-1, which are also known VDAC binding partners impart the same physiological regulation on mitochondrial [Ca²⁺]. Furthermore, the specific physiological consequences and significance of this regulation remain to be determined.Increased production and reduced scavenging of reactive oxygen species (ROS) is frequently observed in cancer cells.¹⁰ While excessive ROS levels are toxic, sub-lethal production serves an important signaling function, particularly in cancers, were ROS promote cell proliferation, migration and invasion.^{11, 12, 13, 14, 15} A primary source of ROS are the mitochondria, and a number of mitochondrial signaling pathways are known to be remodeled and contribute to elevated ROS in cancer cells, including those involved in regulating the electron transport chain (ETC) function and metabolic activity.^{11,16, 17, 18} It is recognized that upregulation of antiapoptotic Bcl-2 proteins are also associated with a pro-oxidant intracellular environment.^{19, 20, 21, 22} Mechanistically, they are thought to act at the level of the mitochondria to affect the respiratory chain and increase production of ROS. Since matrix [Ca²⁺] is an important regulator of mitochondrial metabolism,^23,24 and as such, contributes to the regulation of mitochondrial ROS production,²⁵ we reasoned that antiapoptotic Mcl-1/VDAC interactions could promote ROS generation by facilitating matrix Ca²⁺ uptake.Understanding non-canonical roles of Mcl-1 is an important step toward identifying novel therapeutic targets, particularly in cancers where it is highly expressed, such as in non-small cell lung cancer (NSCLC).^26,27 Therefore, we hypothesized that Mcl-1 binding to VDAC promotes mitochondrial Ca²⁺ uptake and ROS production in NSCLC cells and that this is essential in maintaining the cancer cell phenotype. To test this, we assessed the biochemical interaction between Mcl-1 and VDAC and examined the effects of manipulating Mcl-1 expression levels and Mcl-1/VDAC interactions on mitochondrial Ca²⁺ uptake, ROS generation and NSCLC cell proliferation and migration.     

Elimination of quiescent/slow-proliferating cancer stem cells by Bcl-XL inhibition in non-small cell lung cancer

Lung cancer is the most common cause of cancer-related mortality worldwide, urging the discovery of novel molecular targets and therapeutic strategies. Stem cells have been recently isolated from non-small cell lung cancer (NSCLC), thus allowing the investigation of molecular pathways specifically active in the tumorigenic population. We have found that Bcl-X_L is constantly expressed by lung cancer stem cells (LCSCs) and has a prominent role in regulating LCSC survival. Whereas chemotherapeutic agents were scarcely effective against LCSC, the small molecule Bcl-2/Bcl-X_L inhibitor ABT-737, but not the selective Bcl-2 inhibitor ABT-199, induced LCSC death at nanomolar concentrations. Differently from gemcitabine, which preferentially eliminated proliferating LCSC, ABT-737 had an increased cytotoxic activity in vitro towards quiescent/slow-proliferating LCSC, which expressed high levels of Bcl-X_L. In vivo, ABT-737 as a single agent was able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors. Altogether, these results indicate that quiescent/slow-proliferating LCSC strongly depend on Bcl-X_L for their survival and indicate Bcl-X_L inhibition as a potential therapeutic avenue in NSCLC.Lung cancer is the leading cause of cancer-related death in men and is expected to become the main cause of cancer death for women in the near future.^{1, 2} There is increasing evidence that cancer stem cells (CSCs) have a key role in drug resistance, tumor progression and metastasis in multiple tumor types, including lung cancer.³ Lung cancer stem cells (LCSCs) have been previously identified through different criteria including surface expression of CD133, c-kit or through functional properties such as selective drug survival, elevated aldehyde dehydrogenase (ALDH) activity, increased glycolysis and glycine/serine metabolism or low concentrations of reactive oxygen species and ATP.^{4, 5, 6, 7, 8, 9} Importantly, when inoculated into immunocompromised mice, LCSCs give rise to xenografts that histologically reproduce the tumor of origin, thus representing an improved model for in vivo testing of new targeted therapies.¹⁰ Several tumors express elevated levels of anti-apoptotic Bcl-2 family proteins such as Bcl-2, Bcl-X_L and Mcl-1, which affect the apoptotic threshold of neoplastic cells contributing to chemotherapy resistance.¹¹ Inhibition of anti-apoptotic Bcl-2 family members has been for long time regarded as a promising strategy to induce cancer cell death through approaches of increasing specificity. BH3 mimetics such as ABT-737, the related orally available ABT-263 (navitoclax) and the recently developed Bcl-2-selective inhibitor ABT-199 have been shown to exert an antitumor effect in preclinical and clinical settings either as single agents or in combination with conventional or targeted drugs.¹² Recently, a new role for Bcl-2 has emerged in acute myeloid leukemia (AML), where quiescent stem cells characterized by low levels of reactive oxygen species were found to overexpress Bcl-2 and rely on this factor for survival.¹³ Similarly, in chronic myeloid leukemia (CML), quiescent therapy-resistant stem cells were sensitized to tyrosine kinase inhibitors by treatment with a pan-Bcl-2 inhibitor.¹⁴ In solid tumors, the role of Bcl-2 family members in regulating the stem cell compartment is less clear. By analyzing the expression and relative function of Bcl-2 and Bcl-X_L in LCSC, we identified a prevalent role of Bcl-X_L in LCSC survival. Differently from chemotherapy, ABT-737 showed a preferential cytotoxic activity towards quiescent/slowly proliferating LCSC in vitro indicating a potential use of this inhibitor to eradicate chemotherapy-resistant LCSC. In vivo, ABT-737 blocked the progression of LCSC-derived xenografts and reduced CSC content, substantiating its specific effect on the CSC compartment. Altogether, these results indicate for the first time a key role of Bcl-X_L in LCSC, opening new perspectives for the elimination of therapy-resistant cells.     

Mcl-1 is a key regulator of the ovarian reserve

A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.Estimates of the human primordial follicle (PMF) reservoir, the size of which dictates the extent of the ovarian reserve, indicates the presence of at least half a million oocytes per ovary at birth.^{1, 2} The essential decision that PMFs face is either long-term arrest with a possibility of recruitment toward the growing pool, or death. Even upon recruitment to the growing pool, intricately orchestrated crosstalk of survival signals between ovarian somatic cells and oocytes facilitate the ovulation of a single oocyte in human in each cycle. Hence, the default fate for millions of ovarian germ cells is death, as only a small fraction survive till ovulation.³ Insufficient endowment during fetal development or excessive oocyte loss during postnatal life further limits the ovarian reserve and can result in an untimely exhaustion of the follicle pool leading to premature ovarian failure (POF); a syndrome that affects around 1% of all women, with a higher prevalence (up to 30%) in families with heritable traits of this condition.^{4, 5} Mechanisms responsible for maintenance of the follicular reserve are poorly understood, however, biological assessments and mathematical modeling reveal that progressive loss of follicles with age is non-linear and accelerates, especially after 38 years.^{6, 7} With a declining ovarian reserve, poor oocyte quality is an additional factor that contributes to the reduced fertility associated with increased maternal age. Oocytes and resulting embryos of older mothers have increased rates of aneuploidies likely due to defects in chromosomal cohesion and meiotic spindle stability, decreased DNA repair capacity, altered gene expression, impaired mitochondrial function and elevated cellular redox, all contributing to increased rates of cell death.^{8, 9, 10}The marked decline of oocyte number in mammalian ovaries has been attributed to oocyte loss via stage-specific modes of death. As yet, perinatal PMF loss in mice most frequently engages apoptotic cell death,^{11, 12} whereas within the postnatal ovary, oocytes in growing follicles undergo atresia, a less ''molecularly'' defined death, carrying hallmarks of both apoptosis and autophagy.^{13, 14, 15} It is thus surprising that no member of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family has been identified with a definitive role in governing oocyte survival and the maintenance of the ovarian reserve. Bcl-2l2/Bcl-w and Bcl-2-l10/Diva deficiency had no apparent impact on the ovarian reserve, and although ablation of Bcl-2 led to a loss of one-third of the adult PMF pool, the growing follicle pool was not significantly impacted and these animals did not undergo POF.^{16, 17, 18, 19} Conditional Bcl-x (Bcl-2l1) inactivation led to increased primordial germ cell apoptosis in the embryo,²⁰ but postnatal inactivation of Bcl-x in oocytes did not compromise the ovarian reserve in young females.²¹ Bcl2a1a/Bfl-1/A1 was low to undetectable in fully grown germinal vesicle (GV) or ovulated murine oocytes,²² however, the impact of Bfl-1 deficiency on the ovarian reserve has not yet been analyzed to the best of our knowledge. Consequently, either various anti-apoptotic Bcl-2 members have overlapping roles in governing postnatal oocyte survival and maintenance of the adult ovarian reserve in mice, or the anti-apoptotic Bcl-2 member that regulates this decision has yet to be identified.     

Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP₃ receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP₃Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP₃R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.Lung cancer accounts for 12% of all new cancers worldwide and is a leading cause of cancer-related mortality in the United States.^{1, 2, 3} Although small cell lung cancer (SCLC) comprises only 15% of lung cancer cases,^{2, 3} it has an annual mortality rate approaching that of breast and prostate cancer.⁴ Compared with the more common non-small cell lung cancer (NSCLC), SCLC is more aggressive and associated with rapid development of metastasis.² Moreover, although SCLC is more responsive to chemotherapy and radiation therapy initially, it typically relapses quickly with treatment-resistant disease.² In contrast to dramatic advances in chemotherapy and personalized medicine in other malignancies, the life expectancy of SCLC patients has remained <2 years for decades and is <1 year for patients with extensive disease.^{5, 6} The lethality of SCLC is attributed in part to the development of resistance to standard combination chemotherapies, underscoring the need to develop novel therapeutic approaches based on understanding the molecular and cellular biology of SCLC.^{5, 6}Evasion from apoptosis is a major hallmark of cancer and a prominent factor underlying drug resistance in SCLC.³ Multiple mechanisms contribute to apoptosis resistance in SCLC, including elevated expression of the antiapoptotic Bcl-2 protein³ (Supplementary Figure S1). Tsujimoto and colleagues discovered elevated levels of Bcl-2 mRNA and protein in SCLC cells not long after their identification of Bcl-2 as the protein product of the bcl-2 gene in follicular lymphoma.^{7, 8} Subsequently, immunohistochemistry of 164 primary SCLC samples revealed 76% were positive for Bcl-2, a finding substantiated by microarray detection of increased BCL-2 mRNA levels in 84% of SCLC samples^{9, 10} and by genomic sequencing of circulating SCLC tumor cells.¹¹ Moreover, proteomic profiling documented that Bcl-2 is more highly expressed in SCLC than in NSCLC, reflecting the vastly different biology of these lung cancer subtypes.¹²The major known function of Bcl-2 is to bind and sequester BH3-only proteins such as Bim, preventing these proteins from inducing apoptosis.^{13, 14, 15} Therefore, a major investment has been made in targeting this interaction for cancer treatment. The interaction takes place in a hydrophobic groove on Bcl-2 and the therapeutic strategy for targeting this interaction has been to develop small molecules, BH3-mimetic agents, which bind in the hydrophobic groove and induce apoptosis by displacing the BH3-only proteins. This approach has been reviewed in detail.^{14, 15, 16}Among BH3-mimetic agents advancing through clinical trials for both hematological malignancies^{15, 17} and solid tumors¹⁸ are ABT-737 and its orally bioavailable derivative ABT-263 (Navitoclax). Reported studies of ABT-199, a selective inhibitor of Bcl-2, are at present limited to hematological malignancies.¹⁸ In screening a large number of cancer cell lines, the pioneering work of Oltersdorf et al.¹⁹ demonstrated potent single-agent activity of ABT-737 against cell lines representative of lymphoid malignancies and SCLC. Clinical trials of ABT-263, an orally bioavailable version of ABT-737, achieved overall response rates ranging from as high as 35% in relapsed/refractory chronic lymphocytic leukemia (CLL) and 22% in follicular lymphoma.¹⁷ Reported responses are generally less in solid tumors with the notable exception of SCLC.¹⁸ But even in SCLC, activity of ABT-263 is limited in comparison to hematological malignancies, with 1 of the 39 (3%) of patients achieving a partial response to ABT-263 and 9 of the 37 (23%) achieving stable disease in a phase I clinical trial.²⁰ This experience suggests a need to develop additional ways of targeting Bcl-2 for cancer treatment.A potential alternative therapeutic target for Bcl-2-positive malignancies involves interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP₃R), an IP₃-gated Ca²⁺ channel located on the endoplasmic reticulum (ER). Bcl-2 is located not only on the outer mitochondrial membrane but also on the ER, and at both of these locations, it functions as a potent inhibitor of apoptosis.^{21, 22, 23} ER-localized Bcl-2 interacts with IP₃Rs and inhibits apoptosis by preventing excessive IP₃R-mediated Ca²⁺ transfer from the ER lumen into the cytoplasm and nearby mitochondria.^{24, 25, 26} Notably, regions of Bcl-2 involved in binding BH3-only proteins and IP₃Rs are entirely different. Whereas BH3-only proteins and their BH3-mimetic counterparts bind in a hydrophobic groove composed of BH3 domains 1–3 of Bcl-2,^{13, 14} the BH4 domain of Bcl-2 is necessary for interaction with IP₃Rs.²⁷ To develop a peptide inhibitor of Bcl-2–IP₃R interaction, we identified the Bcl-2-binding region on the IP₃R and developed a small synthetic 20 amino-acid peptide corresponding to this region.²⁸ This peptide, when fused to the cell-penetrating peptide of HIV TAT, binds to the BH4 domain of Bcl-2 and functions as a decoy peptide, inhibiting Bcl-2–IP₃R interaction.^{29, 30} We currently refer to this peptide as BIRD-2 (Bcl-2-IP₃ Receptor Disruptor-2), having formerly named it TAT-IDP_DD/AA.³¹ By disrupting the Bcl-2–IP₃R interaction, BIRD-2 abrogates Bcl-2 control over IP₃R-mediated Ca²⁺ elevation and induces Ca²⁺-mediated apoptosis in primary human CLL cells²⁹ and diffuse large B-cell lymphoma cells.³² Notably, BIRD-2 does not kill normal cells, including human lymphocytes isolated from peripheral blood²⁹ and normal murine embryonic fibroblasts (F Zhong and C Distelhorst, unpublished data).The present investigation was undertaken to determine whether Bcl-2–IP₃R interaction is a potentially useful therapeutic target in SCLC. In support of this concept, we find the majority of SCLC lines tested are sensitive to BIRD-2-induced apoptosis and that BIRD-2 induces apoptosis in several ABT-263-resistant SCLC lines. BIRD-2, we find, lacks generalized cytotoxicity as it does not induce cell death in NSCLC lines or a normal lung epithelial line. On the other hand, we find that BIRD-2 and ABT-263 synergize in killing SCLC cells. These findings for the first time provide preclinical evidence of the potential value of targeting both antiapoptotic mechanisms of Bcl-2 for the treatment of SCLC.     

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.     

Deubiquitylating enzyme USP9x regulates radiosensitivity in glioblastoma cells by Mcl-1-dependent and -independent mechanisms

Glioblastoma is a very aggressive form of brain tumor with limited therapeutic options. Usually, glioblastoma is treated with ionizing radiation (IR) and chemotherapy after surgical removal. However, radiotherapy is frequently unsuccessful, among others owing to resistance mechanisms the tumor cells have developed. Antiapoptotic B-cell leukemia (Bcl)-2 family members can contribute to radioresistance by interfering with apoptosis induction in response to IR. Bcl-2 and the closely related Bcl-xL and Mcl-1 are often overexpressed in glioblastoma cells. In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a short-lived protein whose stability is closely regulated by ubiquitylation-dependent proteasomal degradation. Although ubiquitin ligases facilitate degradation, the deubiquitylating enzyme ubiquitin-specific protease 9x (USP9x) interferes with degradation by removing polyubiquitin chains from Mcl-1, thereby stabilizing this protein. Thus, an inability to downregulate Mcl-1 by enhanced USP9x activity might contribute to radioresistance. Here we analyzed the impact of USP9x on Mcl-1 levels and radiosensitivity in glioblastoma cells. Correlating Mcl-1 and USP9x expressions were significantly higher in human glioblastoma than in astrocytoma. Downregulation of Mcl-1 correlated with apoptosis induction in established glioblastoma cell lines. Although Mcl-1 knockdown by siRNA increased apoptosis induction after irradiation in all glioblastoma cell lines, USP9x knockdown significantly improved radiation-induced apoptosis in one of four cell lines and slightly increased apoptosis in another cell line. In the latter two cell lines, USP9x knockdown also increased radiation-induced clonogenic death. The massive downregulation of Mcl-1 and apoptosis induction in A172 cells transfected with USP9x siRNA shows that the deubiquitinase regulates cell survival by regulating Mcl-1 levels. In contrast, USP9x regulated radiosensitivity in Ln229 cells without affecting Mcl-1 levels. We conclude that USP9x can control survival and radiosensitivity in glioblastoma cells by Mcl-1-dependent and Mcl-1-independent mechanisms.Along with surgery, radiotherapy, and chemotherapy are the main treatment options of tumors. While the former aims to remove the tumor bulk mass, the latter two intend to neutralize remaining tumor cells. Ionizing radiation (IR) exerts its cytotoxic effects by inducing cell death. One form of specific cell death induced by IR is intrinsic apoptosis, which is regulated by members of the B-cell leukemia (Bcl)-2 protein family.¹The Bcl-2 protein family consists of protective antiapoptotic and pro-apoptotic members, which keep each other in check by antagonizing each other''s function.² The activation of pro-apoptotic multidomain proteins Bax and Bak is essential to induce mitochondrial outer membrane permeabilization, resulting in the release of cytochrome C and other apoptotic factors into the cytosol where, in turn, caspases become activated. Antiapoptotic Bcl-2 family members prevent the activation of Bax and Bak either by direct interaction or indirectly by sequestering pro-apoptotic BH3-only proteins Bim and Bid that are required to activate Bax and Bak. Other BH3-only proteins are also able to bind to antiapoptotic proteins, thereby releasing Bax and Bak from their inhibitory complexes with antiapoptotic proteins. Changing the balance between anti- and pro-apoptotic Bcl-2 family members can shift the cells toward survival or apoptosis, depending on whether the protective or the detrimental proteins dominate.Bcl-2 itself, Bcl-xL, and myeloid cell lymphoma-1 (Mcl-1) belong to the antiapoptotic proteins of the Bcl-2 family. They are often overexpressed in tumor cells and are associated with increased resistance to apoptosis induction in response to radio- and chemotherapy.^{3, 4} As more than one of the protective proteins can be upregulated in tumors, the neutralization of all antiapoptotic proteins is needed to successfully induce apoptosis. Blocking the antiapoptotic function of Bcl-2/Bcl-xL by inhibitors mimicking BH3-only proteins, such as ABT737 and ABT263, can induce apoptosis in cells with low Mcl-1 levels but has no effect on cells with high Mcl-1 levels.^{5, 6, 7} In contrast, specific inhibitors targeting Mcl-1 have been insufficiently described until now. However, Mcl-1 availability might be modulated by targeting pathways that regulate Mcl-1 stability.In contrast to Bcl-2 and Bcl-xL, Mcl-1 is a relatively short-lived protein.^{8, 9} Usually, Mcl-1 is quickly ubiquitylated by specific ubiquitin ligases and targeted for proteasomal degradation. Phosphorylation of Mcl-1, for example by glycogen synthase kinase GSK-3β, can accelerate this degrading process,^{10, 11} whereas deubiquitinases counteract it by removing the polyubiquitin chain, thereby stabilizing the short-lived protein. The ubiquitin-specific protease 9x (USP9x) was recently identified as a Mcl-1 specific deubiquitinase.¹² However, the circumstances under which USP9x regulates Mcl-1 stability are not well understood. Schwickart et al.¹² showed that USP9x levels correlated with Mcl-1 levels, suggesting a constitutive regulation of Mcl-1 levels by the deubiquitinase. In contrast, our recent results showed no effect of USP9x on Mcl-1 levels in healthy Jurkat cells, but an accelerated IR-induced Mcl-1 degradation was detected when USP9x was knocked down.⁹ This indicates that the association of USP9x with Mcl-1 is regulated by a yet unknown mechanism in response to irradiation.In the present study, we aimed to analyze the impact of USP9x on Mcl-1 and cell survival in glioblastoma cell lines. Glioblastoma is not only the most common but also a very aggressive form of brain tumor that are primarily removed by surgery as radically as possible and consecutively treated with radiochemotherapy, if the patient''s condition allows for adjuvant therapy.¹³ Despite the multimodal treatment, the median patient survival is below 1.5 years. Comparing human grade III astrocytoma with grade IV glioblastoma samples, we could show that Mcl-1 and USP9x are upregulated during tumor progression. Furthermore, we examined four established (A172, U373, Ln229, T98G) and two primary (LKI, WKI) glioblastoma cell lines that differ in their ability to downregulate Mcl-1 and induce apoptosis in response to IR. Analyzing A172 and U373 cells more closely, we detected an increased Mcl-1 ubiquitylation that correlated with a reduced Mcl-1 stability 48 h after irradiation in U373 cells, but not in A172 cells. Moreover, Mcl-1 knockdown sensitized A172, Ln229, and T98G cells to IR-induced apoptosis, suggesting that Mcl-1 is an important factor increasing glioblastoma cell survival after irradiation. In contrast, USP9x knockdown slightly increased apoptosis in IR-resistant A172 cells and significantly in Ln229 cells and reduced clonogenic survival after irradiation only on these two cell lines. Although USP9x knockdown reduced Mcl-1 levels and increased apoptosis in A172 cells, USP9x regulated radiosensitivity independently of Mcl-1 in Ln229 cells.Our results show a different requirement of USP9x in the control of glioblastoma cell survival and radiosensitivity.     

Nanoencapsulation of ABT-737 and camptothecin enhances their clinical potential through synergistic antitumor effects and reduction of systemic toxicity

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.Colorectal cancer is the third most commonly occurring cancer worldwide. Despite advances in understanding the molecular basis of the disease and development of new therapeutic modalities, the 5-year overall survival for patients with late-stage disease remains poor.¹ Although new compounds are continually developed, the lack of efficacy at systemically tolerable doses frequently precludes their success in the clinic. Formulation and delivery strategies that can improve the narrow therapeutic window of such compounds have the potential to make significant advances in treatment regimens.ABT-737 is a small molecule that targets the BH3-binding hydrophobic cleft of antiapoptotic B-cell lymphoma (Bcl) proteins Bcl-2, Bcl-w and Bcl-X(L), which are frequently upregulated in tumors and strongly associated with chemoresistance.² As a single agent, ABT-737 is particularly potent against leukaemia/lymphoma cells and a range of small-cell lung carcinomas, causing complete regression of solid tumors in mouse models.³ Moreover, its orally bioavailable derivative, ABT-263, has shown potential clinical utility in combination therapies as it has been demonstrated to sensitize tumor types, including colorectal cancer cells, to a range of chemotherapies.⁴ However, the clinical evaluation of ABT-263 has revealed that its therapeutic effects are compromised by severe dose-dependent thrombocytopenia.^{5, 6, 7} Platelets normally survive in circulation for several days by maintaining elevated levels of Bcl-X(L), the inhibition of which by ABT-263/ABT-737 results in premature initiation of platelet apoptosis.^8,9 In spite of these issues, ABT-263 is still under clinical investigation in combination with frontline cytotoxic chemotherapies such as irinotecan and other therapies (Supplementary Table S1).Camptothecin (CPT) is a potent topoisomerase-I inhibitor; however, it is poorly soluble and rapidly hydrolyzed from a lactone to an inactive carboxylic acid in an aqueous environment, thus limiting its clinical applicability. Consequently, this has led to the development of derivatives such as irinotecan.¹⁰ Although irinotecan overcomes some of the pharmacokinetic problems associated with CPT, it has reduced inhibitory activity and still exhibits systemic toxicities such as neutropenia and dose-limiting diarrhea owing to the damage of the intestinal mucosa.¹¹Pharmaceutical formulations that improve the safety profile of potent anticancer drugs such as ABT-737/263 and CPT are urgently required. Drug-loaded nanotherapeutics are finding increasing application in a range of solid tumors, best exemplified by Doxil, a liposomal preparation of doxorubicin that reduces drug-associated myocardiotoxicity.¹² Such drug carriers can not only diminish adverse effects, but also simultaneously enhance tumor localization through the ability of nanosized particles to penetrate defective endothelial junctions in the tumor neovasculature – a phenomenon known as the enhanced permeability and retention (EPR) effect.¹³ Of note, a polymeric formulation of CPT, CRLX101, has been clinically evaluated, and it was demonstrated that the improved pharmacokinetics and tumor penetration of nanoparticle (NP)-based carriers observed in mouse models were maintained in patients,¹⁴ highlighting the therapeutic potential that new formulations hold for this parental molecule.Previously, we developed polymeric NPs encapsulating a range of drug types, formulated in FDA-approved poly-lactide-co-glycolide acid (PLGA).^{15, 16, 17} In the current investigation, we wished to examine the application of both CPT and ABT-737 in colorectal cancer models and determine if a nanodelivery system could be employed to elicit the synergistic efficacies of these agents. We show that nanoencapsulation of ABT-737 reduces thrombocytopenic effects, whereas nanoencapsulation of CPT inhibits its cytotoxic effects towards white blood cells and the gastrointestinal (GI) tract. Both drugs were successfully combined in a single NP formulation to elicit synergistic effects against colorectal cancer cells in vitro and in vivo; this highlights the potential of this approach and similar formulations for widening narrow therapeutic windows for the treatment of colorectal and other cancers with rationally selected drug combinations delivered at pre-determined synergistic concentrations.     

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.     

Radiosensitization by a novel Bcl-2 and Bcl-XL inhibitor S44563 in small-cell lung cancer

Radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. In the current study, we investigated whether using the Bcl-2/Bcl-X_L inhibitor S44563 can enhance radiosensitivity of SCLC cells in vitro and in vivo. In vitro studies confirmed that S44563 caused SCLC cells to acquire hallmarks of apoptosis. S44563 markedly enhanced the sensitivity of SCLC cells to radiation, as determined by a clonogenic assay. The combination of S44563 and cisplatin-based chemo-radiation showed a significant tumor growth delay and increased overall survival in mouse xenograft models. This positive interaction was greater when S44563 was given after the completion of the radiation, which might be explained by the radiation-induced overexpression of anti-apoptotic proteins secondary to activation of the NF-κB pathway. These data underline the possibility of combining IR and Bcl-2/Bcl-X_L inhibition in the treatment of SCLC as they underscore the importance of administering conventional and targeted therapies in an optimal sequence.Identifying the mechanisms leading to radioresistance including resistance to apoptosis is essential to improve clinical outcome in cancer patients. Disabled apoptosis has been catalogued among the fundamental hallmarks of cancer¹ and the proteins of the Bcl-2 family play a fundamental role in regulating this modality of cell death. The Bcl-2 family comprises both pro- and anti-apoptotic members; the latter (Bcl-2, Bcl-X_L and Mcl-1) are often overexpressed in cancer cells to facilitate the survival of cells that under normal circumstances should have undergone apoptosis.² The molecular interactions between pro- and anti-apoptotic Bcl-2 family members determine cellular sensitivity to multiple lethal triggers, including many standard chemotherapeutic agents and ionizing radiation (IR).^{3, 4} Overexpression of Bcl-2 is known to increase clonogenic survival and inhibit IR-induced apoptosis.^{3, 4} Bcl-X_L expression also shows a strong correlation with resistance to cytotoxic anticancer therapies including IR.^{5, 6}Lung cancer is the leading cause of cancer deaths in western countries.⁷ Small-cell lung cancer (SCLC) accounts for 15% of all lung cancer cases and is distinguished from non-SCLC by its characteristic cytomorphology, rapid proliferation and early dissemination to metastatic sites.⁸ The standard of care to patients with limited-stage SCLC and good performance status is based on a combination of IR and cisplatin-based chemotherapy, resulting in a complete response rate as high as 50–80% coupled to a deceptive 12–20% 5-year survival.⁹ Initially, SCLC is responsive to chemo- and radiotherapy. However, SCLC recurs within the first 12 months.¹⁰ To date, the pathways mediating chemo- and radioresistance in SCLC are largely unknown.Deletion of pro-apoptotic gene and amplification of anti-apoptotic gene are frequently observed in SCLC, especially amplification of the BCL2L1 and BCL2L2 genes.¹¹ At the protein level, increased expression of Bcl-2 has been reported in up to 90% of metastatic SCLC. Bcl-2 overexpression, downregulation of the pro-apoptotic Bcl-2 antagonist Bax and a shift in the Bcl-2/Bax ratio to levels >1 are correlated with lower apoptotic index in tumors¹² and are associated with chemotherapeutic resistance in SCLC cell lines.¹³ In contrast with most solid tumor cell lines, where apoptosis does not appear as a predominant cell death mechanism after IR,¹⁴ overexpression of Bcl-2 can abrogate chemotherapy-induced apoptosis in SCLC cell lines.¹³ Apoptosis may be one of the mechanisms that cause SCLC cells to die in response to radiotherapy.^{15, 16}Recently, a small synthetic compound ABT-737 and its orally bioavailable form ABT-263 (Navitoclax) were shown to efficiently antagonize Bcl-2 and Bcl-X_L by binding to their BH3 receptor domain. ABT737 or its derivatives mediate antitumoral effects in chronic lymphocytic leukemia (CLL) and SCLC in preclinical and early clinical trials.^{17, 18} However, there is no published study that evaluates the combination of new Bcl-2/Bcl-X_L inhibitors, IR and chemo-radiotherapy.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.