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.     

RILP suppresses invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS

RILP (Rab7-interacting lysosomal protein) is a key regulator for late endosomal/lysosomal trafficking, and probably a tumor suppressor in prostate cancer. However, the role of RILP in other cancers and the underlying mechanism for RILP in regulating the invasion of cancer cells remain to be investigated. In this study, we showed that overexpression of RILP in breast cancer cells inhibits the migration and invasion, whereas the depletion of RILP by RNAi-mediated knockdown promotes the migration and invasion. We identified RalGDS (Ral guanine nucleotide dissociation stimulator) as a novel interacting partner for RILP, and truncation analysis revealed the N-terminal region of RILP is responsible for interacting with the guanine nucleotide exchange factor (GEF) domain of RalGDS. Immunofluorescence microscopy revealed that RalGDS can be recruited to the late endosomal compartments by RILP. Further investigations indicated that the overexpression of RILP inhibits the activity of RalA, a downstream target of RalGDS. Our data suggest that RILP suppresses the invasion of breast cancer cells by interacting with RalGDS to inhibit its GEF activity for RalA.Diverse alternations of oncogenic factors can either activate or inactivate signaling pathways involved in cell proliferation, migration and apoptosis that are intimately associated with cancer development.^{1, 2, 3} Recent studies suggest that the derailed membrane trafficking is also closely related to cancer development. Activation or attenuation of signal transduction is usually linked to membrane trafficking. The recycling and degradation of surface receptors, such as EGFR, will influence downstream signaling pathways.^{4, 5} Therefore, the cross-talk between membrane trafficking and signaling pathway could be the novel mechanism associated with cancer development.Alternations of the membrane trafficking machineries are established as the causes for some cancers. For examples, Rab25 is overexpressed in breast and ovary caners,⁶ and recent investigations suggest that Rab25 is also related to other cancers.^{7, 8, 9} Arf6 is a vital regulator for the invasive activity of breast cancer cells.¹⁰ Disordered membrane trafficking is emerging as an important property during tumorigenesis, thus the membrane trafficking machineries are potential therapeutic targets for cancer treatment.Rab small GTPases are considered as the master regulators for membrane trafficking.¹¹ The interactions between Rab proteins and their downstream effectors are involved in various steps of vesicle trafficking such as tethering and fusion. Aberrant activities of Rab proteins are closely related to some cancers.^{12, 13, 14, 15} Some Rab proteins mediate the trafficking of cargos, especially membrane proteins on the plasma membrane, such as integrin and E-cadherin. Their aberrant trafficking is proposed to be the underlying mechanism for the functional regulation of Rab protein in cancer cells.^{16, 17}Rab7, together with its downstream effector RILP (Rab7-interacting lysosomal protein), are the key regulators for late endosomal/lysosomal trafficking. RILP interacts with activated GTP-bound Rab7 through its carboxylic terminal region, whereas interacting with dynein/dynactin complex is mediated through its amino region, driving late endosomal/lysosomal trafficking, especially lysosomal positioning.^{18, 19} Rab7 has been demonstrated to be an important factor for cell growth and survival.^{20, 21} Recently, Steffan et al.²² found that RILP suppresses the invasion of prostate cancer cells through inhibiting the anterograde trafficking of lysosomes.²³ Whether the potential role of Rab7-RILP in cell migration/invasion is also implicated in other cancers is of interest to investigate and the underlying molecular mechanism is yet to be defined.In this study, we found that RILP suppresses the proliferation, migration and invasion of breast cancer cells. We also identified (Ral guanine nucleotide dissociation stimulator (RalGDS) as a novel interacting partner for RILP. The interaction of RILP with RalGDS modulates the activity of RalA. Our results suggest that RILP suppresses the invasion of breast cancer cells by modulating the activity of RalA through interaction with RalGDS.     

Daytime Blue Light Enhances the Nighttime Circadian Melatonin Inhibition of Human Prostate Cancer Growth

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462–484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake–metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.Abbreviations: A-V, arterial–venous difference, ipRGC, intrinsically photosensitive retinal ganglion cell, LA, linoleic acid, 13-HODE, 13-hydroxyoctadecadienoic acid, TFA, total fatty acidsLight profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in all mammals and is essential to life on our planet.^{2,15,28, 40} The light–dark cycle entrains the master biologic clock, located in the suprachiasmatic nucleus of the brain, in an intensity-, duration-, and wavelength-dependent manner.^8-13 Photobiologic responses, including circadian rhythms of metabolism and physiology, are mediated by organic molecules called ‘chromophores,’ which are contained within a small subset of retinal cells, called the intrinsically sensitive retinal ganglion cells (ipRGC).^{16,29,31,36,41,49,53,59} In humans and rodents light quanta are detected by the chromophore melanopsin, which detects light quanta in principally the short-wavelength, blue-appearing portion of the spectrum (446 to 477 nm), and transmits its photic information via the retinohypothalamic tract to the ‘molecular clock’ of the suprachiasmatic nucleus. This region of the brain regulates the daily pineal gland production of the circadian neurohormone melatonin (N-acetyl-5-methoxytryptamine), which results in high levels produced at night and low levels during daytime.^38,54 The daily, rhythmic melatonin signal provides temporal coordination of normal behavioral and physiologic functions including chronobiologic rhythms of locomotor activity,² sleep-wake cycle,^2,14 dietary and water intake,^2,51 hormone secretion and metabolism.^5,44,47,61 Alterations in light intensity, duration, and spectral quality at a given time of day,^{8-13,17,19-22,24,61} such as occurs in night-shift workers exposed to light at night,^26,34,46,57 acutely suppresses endogenous melatonin levels in most mammalian species^{9,11,44,45,54,55} and may lead to various disease states, including metabolic syndrome^5,61 and carcinogenesis.^4-7,17,18Recent studies from our laboratory^{5,20,23-25,60,61} have demonstrated that relatively small changes in the spectral transmittance (color) of light passing through translucent amber (>590 nm), blue (>480 nm), and red-tinted (>640 nm) polycarbonate laboratory rodent cages, compared with standard polycarbonate clear cages (390 to 700 nm), during the light phase markedly influenced the normal nighttime melatonin signal and disrupted temporal coordination of metabolism and physiology.^19,24,61 Most notable was our discovery that, in both male and female pigmented nude rats maintained in blue-tinted rodent cages, nighttime melatonin levels were as much as 7 times higher than normal nighttime peak levels in animals maintained in all other cage types.¹⁹ An earlier study in human subjects diagnosed with midwinter insomnia coupled with low nighttime melatonin levels demonstrated that daily exposure to intense morning bright polychromatic light therapy for up to one week resulted in a restoration of nocturnal melatonin levels to those of control subjects.³⁵ In another study, exposure to blue-tinted (470 nm) LED light (100 lx) for approximately 20 min in the morning after 2 sleep-restricted (6 h) nights led to earlier onset of the melatonin surge at nighttime.³⁰In the United States alone this year, approximately 240,000 men will be diagnosed with prostate cancer, and nearly 30,000 will die from this disease (National Cancer Institute; www.cancer.gov/). Epidemiologic studies have shown that night shift work, which involves circadian disruption, including nocturnal melatonin suppression, markedly increases prostate cancer risk in men.^{26,34,46,57,58} Both in vitro and in vivo studies have demonstrated that melatonin inhibits human prostate cancer growth, including that of androgen-receptor–negative, castration-resistant PC3 human prostate cancer cells.^20,29,42,56 Cancer cells depend primarily on aerobic glycolysis (Warburg effect) over oxidative phosphorylation to meet their bioenergetic needs supporting biomass formation.⁵ The Warburg effect is characterized by increased cellular uptake of glucose and production of lactate despite an abundance of oxygen. Investigations have shown that signal transduction pathways that include AKT, MEK, NFκB, GS3Kβ, and PDK1 drive the Warburg effect.^5,61 In addition, cancer cells rely on increased uptake of the ω6 fatty acid linoleic acid (LA), which is prevalent in the western diet.^4-6 In most cancers, LA uptake occurs through a cAMP-dependent transport mechanism, and LA is metabolized to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). In most tumors, 13-HODE plays an important role in enhancing downstream phosphorylation of ERK 1/2, AKT, and activation of the Warburg effect, thereby leading to increased cell proliferation and tumor growth.^4-6 Melatonin, the principal neurohormone of the pineal gland and whose production is regulated by the suprachiasmatic nucleus,^4,5 modulates processes of tumor initiation, progression, and growth in vivo.⁵ The circadian nocturnal melatonin signal not only inhibits LA uptake and metabolism, the Warburg effect in human cancer xenografts, and ultimately tumor growth, but it actually drives circadian rhythms in tumor metabolism, signal transduction activity, and cell proliferation. These effects are extinguished when melatonin production is suppressed by light exposure at night.⁵In the present investigation, we examined the hypothesis that the spectral transmittance (color) of short-wavelength (480 nm) bright light passing through blue-tinted standard laboratory rodent cages during the light phase not only amplifies the normal circadian nocturnal melatonin signal but also enhances the inhibition of the metabolism, signaling activity, and growth progression of human PC3 androgen-receptor–negative human prostate cancer xenografts in male nude rats.     

Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin–proteasome pathway

The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin–proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C^CDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.Inhibitors of microtubule polymerization or depolymerization such as Vinca alkaloids and taxanes, respectively, are widely used as anti-cancer drugs. They arrest cancer cells, inducing mitotic catastrophe and cancer cell death. However, these drugs also affect microtubule function in non-dividing cells and have serious side effects, such as peripheral neuropathy, which limit their utility.¹ Recently, inhibitors of spindle-regulatory proteins, such as mitotic kinases (Aurora kinases and Polo-like kinases) and a motor protein (Eg5/Ksp) have attracted considerable attention, but they have not been developed clinical use yet.^{2, 3}Transforming acidic coiled-coil-3 (TACC3) is another spindle-regulatory protein.^{4, 5} During mitosis, TACC3 localizes to the mitotic spindle and has a critical role in spindle assembly, chromosomal function and mitotic progression.^{6, 7, 8, 9, 10, 11} Studies using microarray and immunohistochemical analysis showed that TACC3 is overexpressed in many human cancers, including ovarian cancer, breast cancer, squamous cell carcinoma and lymphoma.^{12, 13, 14} Depletion of TACC3 results in chromosome alignment defects, multi-polar spindle formation, mitotic cell death and/or a postmitotic cell cycle arrest.^{15, 16, 17, 18, 19, 20} Additionally, conditional disruption of TACC3 has been shown to regress thymic lymphomas in p53-deficient mice without inducing any overt abnormalities in normal tissues.²¹ These findings suggest that TACC3 is a molecular target for anti-cancer drug discovery.The development of a strategy for the selective degradation may be a useful approach to the discovery of novel drugs. Based on the ubiquitin–proteasome system (UPS), we have devised a protein knockdown system for inducing the selective degradation of target proteins by using specifically designed hybrid small compounds.^{22, 23, 24, 25, 26, 27, 28, 29} These compounds, which we have termed SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser), are composed of two different ligands connected by a linker; one is a ligand for cellular inhibitor of apoptosis protein 1 (cIAP1) and the other a ligand for the target protein. Accordingly, SNIPER is expected to crosslink the ubiquitin–ligase cIAP1 and the target protein in the cells, thereby inducing ubiquitylation and, ultimately, proteasomal degradation of the target protein. To date, we have constructed SNIPERs that target cellular retinoic acid binding protein-II (CRABP-II) and nuclear receptors such as estrogen receptor α (ERα) for degradation.^{22, 23, 24, 25, 26, 27, 28} In this study, we designed and synthesized novel SNIPERs targeting TACC3, that is, SNIPER(TACC3)s, that induce proteasomal degradation of the TACC3 protein. We also show that cancer cells expressing a large amount of the TACC3 protein readily undergo cell death as the result of SNIPER(TACC3) treatment.     

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.     

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.