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.     

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.     

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.     

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.     

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21^WAF1/CIP1 in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.Lung cancer is the leading cause of cancer death worldwide, resulting in >1.4 million deaths/year.¹ Lung tumors are often discovered as locally advanced or metastatic disease, and despite improvements in molecular diagnosis and targeted therapies, the overall 5-year survival rate remains in the 10–20% range. Indeed, nonsmall cell lung cancer (NSCLC) is poorly chemosensitive to most of the available agents with response rates ranging from 10 to 25%.² The discovery of recurrent mutations in the epidermal growth factor receptor (EGFR) kinase,³ as well as gene fusion products involving the anaplastic lymphoma kinase (ALK),⁴ has led to a marked change in the treatment of patients with lung adenocarcinoma, the most common type of lung cancer.^{5, 6} To date, patients with mutations in the EGFR gene, suitable for targeting by EGFR tyrosine kinase inhibitors, represent roughly 10%, whereas the subgroup of tumors with ALK rearrangements, targeted by ALK inhibitors, is only ~5%.⁷ Thus, the majority of lung tumors lack effective treatment and novel therapeutic strategies are still needed.MicroRNAs (miRNAs) are short noncoding RNAs, 20–24 nucleotides long, that have important roles in almost all biological pathways,^{8, 9, 10, 11} and influence cancer-relevant processes, such as proliferation,¹² cell cycle,¹³ apoptosis,¹⁴ and migration.¹⁵ Many studies have reported the critical role of miRNAs in lung cancer pathogenesis and their potential as biomarkers for lung cancer risk stratification,¹⁶ outcome prediction,¹⁷ and classification of histological subtypes.^{18, 19} miRNAs are actively released by various cell types and can be detected in biological fluids, such as plasma and serum, making them suitable as circulating biomarkers in NSCLC.^{20, 21}There is limited evidence of mir-660 deregulation in cancer and little is known about its role in lung tumorigenesis and its putative target genes. Mir-660 has been reported to be upregulated in chronic lymphocytic leukemia^{22, 23} and in leukemic cells after treatment with 4-hydroxynonenal, a compound that induces differentiation and blocks proliferation of leukemic cells.²⁴ In a previous study we demonstrated that mir-660 was one of the 24 miRNAs deregulated in plasma samples of NSCLC patients identified in a low-dose computed tomography (LDCT) screening trial.²⁰The p53 tumor suppressor protein is a key regulator of cell cycle G0/G1 checkpoint, senescence, and apoptosis in response to cellular stress signals.^{25, 26} Mouse double minute 2 (MDM2), a p53–E3 ubiquitin ligase,²⁷ is the principal negative regulator of the expression level and function of p53.^{28, 29} Several studies have illustrated different mechanisms of p53 regulation by MDM2,^{30, 31} such as binding transactivation region of p53,^{32, 33} promoting nuclear export and cytoplasmic accumulation of p53 by monoubiquitination,^{34, 35} and inducing p53 proteosomal degradation by polyubiquitination.³⁶ In addition, MDM2 gene has been reported to be amplified or overexpressed in a variety of human cancers, such as sarcoma,³⁷ lymphoma,³⁸ breast cancer,³⁹ lung cancer,⁴⁰ and testicular germ cell tumor.⁴¹ Several miRNAs targeting MDM2 have been identified, such as the mir-143/mir-145 cluster that can be induced by p53,⁴² as well as mir-25 and mir-32, known to inhibit tumor glioblastoma growth in mouse brain.⁴³In this study, we report that mir-660 is downregulated in tissue and plasma samples of lung cancer patients and demonstrate that mir-660 replacement impairs the functionality of p53 wild-type (wt) lung cancer cells and inhibits in vitro and in vivo tumor growth. We showed that all the effects observed after mir-660 overexpression were absent in p53 ko cells, identified MDM2 as mir-660 direct target gene and indicate impairment of the MDM2/p53 interaction as the mechanism underlying tumor growth inhibition.