Akt, FoxO and regulation of apoptosis

In the past decade, it became apparent that immune mediated cell death in a number of autoimmune endocrine diseases was due to the induction of apoptosis in target organ cells. This was conclusively demonstrated for thyroid follicular cells in Hashimoto’s (destructive autoimmune) thyroiditis, but the mechanisms underlying this cell death were not clear. Several hypotheses were put forth involving the role of deathsignalling molecules expressed on thyroid cells. While many of these hypotheses did not hold up under close scrutiny, this stimulated work on the molecular mechanisms of thyroid destruction. Several apoptosis signalling pathways, initiated by molecules such as Fas ligand (FASL) and tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. In this review we will attempt to sort out the inconsistencies in published data on the mechanisms of death-receptor mediated thyroid destruction. We will also review recently proposed models of these mechanisms, and outline directions for research that we feel might lead to discoveries of benefit to the clinician in the treatment and prevention of destructive autoimmune thyroiditis.

Pro-inflammatory sebocyte growth and survival signalling in acne vulgaris are reversed by pro-apoptotic isotretinoin signalling.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance. Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses. Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins. Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211). Citation Format: Margot de Looff, Steven de Jong, Frank A.E. Kruyt. The role of Src in TRAIL signaling in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4377.

In 1995, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was identified based on its sequence homology to other TNF family members (). Among members of the TNF family, TRAIL shares the highest sequence homology with Fas ligand (FasL, CD95L). However, unlike FasL, TRAIL appears to induce apoptosis of tumor cells but not most normal cells (). To date, five receptors for TRAIL have been cloned: TRAIL-R1 (DR4, Apo2A) (), TRAIL-R2 (DR5, TRICK, Killer) (, , , , , ), TRAIL-R3 (DcR1, TRID, LIT) (, , , ), TRAIL-R4 (DcR2, TRUNDD) (), and osteoprotegerin (OPG) (). Unlike other TRAIL receptors, which bind only to TRAIL, osteoprotegerin also binds to osteoprotegerin ligand (OPGL), TRANCE, and RANK ligand (RANKL). TRAIL-R1 and TRAIL-R2 contain intracellular death domains and induce, via coupling with intracellular adaptor proteins, the proteolytic cleavage of caspase-8 (). Caspase-8 activation initiates the extrinsic and intrinsic apoptotic pathways, resulting in caspase-3 cleavage, which is an irreversible step in a cell’s commitment to apoptosis. Other TRAIL receptors do not generate death signals because TRAIL-R3 does not contain a death domain and is attached to the membrane by a glycolipid anchor (,), whereas the death domain of TRAIL-R4 is not functional () and OPG exists only in a soluble form (). Therefore, TRAIL-R3 and TRAIL-R4 might act as decoy receptors by competing with other TRAIL receptors for TRAIL. The expression pattern of TRAIL receptors on certain cell lines might determine their sensitivity to TRAIL. However, the expression of TRAIL decoy receptors is not always related to a cell’s resistance to TRAIL-induced apoptosis (). Other factors may, therefore, play more decisive roles in determining a cell’s sensitivity to TRAIL. In this review, we will focus on NF-κB and PPAR-γ, two transcription factors that were recently found to play important roles in TRAIL-induced apoptosis.

In the present study, it was found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R2 protein expression did not correlate with mRNA expression in melanoma cell lines. In particular, early passage primary cultures from patients had low TRAIL-R2 protein expression compared with later passage cultures although TRAIL-R2 mRNA expression was similar in early and late passages. Similarly, cell lines made resistant to TRAIL by cultures in TRAIL had low TRAIL-R2 protein expression but normal levels of mRNA for TRAIL-R2. Expression from a luciferase reporter gene construct with the 3'-untranslated region (UTR) (but not the 5'-UTR) of TRAIL-R2 was suppressed when transfected into the TRAIL-selected (resistant) melanoma lines compared with that seen in the parental (sensitive) lines. Similar results were seen in early passage (resistant) cultures compared with late passage (sensitive) primary melanoma cultures. RNA gel shift assays demonstrated protein(s) binding to the 3'-UTR of TRAIL-R2 mRNA that were more evident in TRAIL-resistant cultures with low TRAIL-R2 protein expression. A 23-base fragment of the 3'-UTR inhibited binding of the proteins to the 3'-UTR, and a probe using this fragment bound to proteins in TRAIL-selected melanoma lines and early passage isolates of melanoma. Binding of the 3'-UTR probe to the cytosolic protein(s) was induced by exposure to TRAIL and was lost from the TRAIL selected lines 2-3 days after withdrawal of TRAIL from the cultures. These results are consistent with post-transcriptional regulation of TRAIL-R2 expression by cytosolic proteins induced by TRAIL that bind to the 3'-UTR region of TRAIL-R2 mRNA.

We investigated whether snake venom toxin (SVT) from Vipera lebetina turanica enhances the apoptosis ability of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in cancer cells. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29, A549 and HepG2 cells with an even higher dose of TRAIL. SVT, but not TRAIL enhanced expression of cell death receptor (DR) in TRAIL resistant cancer cells in a dose-dependent manner. A combination of SVT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29, A549 and HepG2 cells. Consistent with cell growth inhibition, the expression of TRAIL receptors; DR4 and DR5 was significantly increased as well as apoptosis related proteins such as cleaved caspase-3, -8, -9 and Bax. However, the expression of survival proteins (e.g., cFLIP, survivin, XIAP and Bcl2) was suppressed by the combination treatment of SVT and TRAIL. Depletion of DR4 or DR5 by small interfering RNA significantly reversed the cell growth inhibitory and apoptosis blocking effects of SVT in HCT116 and HT-29 cells. Pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the reactive oxygen species (ROS) scavenger N-acetylcysteine reduced the SVT and TRAIL-induced upregulation of DR4 and DR5 expression, expression of the apoptosis related protein such as caspase-3 and-9, as well as cell growth inhibitory effects. The collective results suggest that SVT facilitates TRAIL-induced apoptosis in cancer cells through up-regulation of the TRAIL receptors; DR4 and DR5 via ROS/JNK pathway signals.

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

Cancer is a widespread disease characterized by unregulated cell growth and evasion of apoptotic stimuli. Most conventional therapies lack cancer cell specificity and induce cell death indirectly through cellular damage in a p53-dependent manner. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). Recombinant human TRAIL (rhTRAIL) can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). CK2 is upregulated in the nucleus of all cancer cells and promotes the expression of inhibitors of apoptosis resulting in resistance to cell death. Therefore, by inhibiting CK2, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. We utilized cotreatment with Ellagic Acid (EA), a natural CK2 inhibitor, and TRAIL against the malignant melanoma cell line A375, which is resistant to TRAIL. Our in vitro analysis showed that TRAIL and EA, as single-agents, can inhibit cell growth in a dose-dependent manner, and the combination treatment of TRAIL plus EA synergistically enhanced the cytostatic effects of the single-agent treatments. We show that TRAIL reduces the viability of A375 cells; whereas, EA although it can inhibit growth, cannot reduce the viability of A375 as determined by Trypan Blue exclusion assays. In combination treatments, the cytotoxic effects of TRAIL were not enhanced. Annexin-V Apoptosis assays and Western Blot analysis show that TRAIL can induce apoptosis in A375 cells characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. Additionally, TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8 activation and the mitochondrial release of cytochrome c, respectively. EA treatment alone did not induce apoptosis and did not induce formation of Annexin-V positive cells or PARP fragmentation. Combination treatments show that EA does not potentiate TRAIL-induced apoptosis. In vivo, TRAIL-treated nude mice bearing A375 xenografts exhibited ∼80% tumor growth inhibition and a prolonged survival rate compared to the untreated control group. EA was not able to inhibit the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA. Citation Format: Katherine Turner, Daniel Lindner, Michael Kalafatis. Effects of TRAIL and ellagic acid on malignant melanoma A375 in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2289. doi:10.1158/1538-7445.AM2014-2289

Objective To explore the effect of chloroquine on death receptor 5 (DR5) expression of hepatocellular carcinoma Huh7 cells and cell proliferation and apoptosis induced by tumor necrosis factor related apoptosis-inducing ligand (TRAIL). Methods Huh7 cells were divided into four groups: the control group (1:1 000 dimethyl sulfoxide), TRAIL group (50 μg/L), chloroquine group (10 μmol/L) and TRAIL+ chloroquine group (TRAIL 50 μg/L+ chloroquine 10 μmol/L). Thiazolyl blue tetrazolium bromide (MTT) assay was used to determine the proliferation activity of cells, immunofluorescence was used to detect the expression of DR5, 4', 6-diamidino-2-phenylindole (DAPI) staining was used to observe cell apoptosis and Western blot was used to detect the expression of cleaved poly ADP-ribose polymerase (PARP). Results TRAIL treatment could decrease Huh7 cells proliferation activity; when compared with the cell viability in the control group, the cell proliferation inhibition rate of chloroquine group, TRAIL group and TRAIL+ chloroquine group was (89±8)%, (53±10)% and (27±7)%, respectively; compared with TRAIL group alone, cell proliferation activity was decreased in TRAIL+ chloroquine group (t= 3.922, P= 0.017). The expression of DR5 was upregulated in chloroquine group, and the cell apoptosis signaling was activated in TRAIL+ chloroquine group. The cell apoptosis rate of TRAIL group and TRAIL+ chloroquine group was (10.0±2.3)% and (20.4±4.0)%, respectively, and there was a statistical difference (t= 3.894, P= 0.018). Conclusion Chloroquine can enhance the cell chemosensitivity to TRAIL treatment by upregulating the expression of DR5 in Huh7 cells. Key words: Carcinoma, hepatocellular; Chloroquine; Death receptor 5; Tumor necrosis factor-related apoptosis inducing ligand

Combination therapy utilizing of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. TRAIL inhibited HCT116 cell growth in a dose-dependent manner; however, this reduction did not occur in TRAIL resistant HT-29 cells with an even higher dose of TRAIL. A combination of PT with TRAIL significantly inhibited cell growth of TRAIL resistant HT-29 cells. Consistent with cell growth inhibition, apoptotic cell death was significantly increased by a combination of PT with TRAIL in both of HCT116 and HT-29 cells. Results of flow cytometry analysis demonstrated that TRAIL-sensitive HCT116 cells had much higher death receptor (DR) 5 than TRAIL-resistant HT-29 cells. Interestingly, treatment of PT and/or TRAIL did not affect DR4/DR5, these results indicate that the apoptotic effect of combination is death receptor-independent apoptosis. We observed that the synergistic effect was associated with Bcl-2 family members, p53 and cytochrome C. Moreover, activation of caspase -3, -8 and -9 was increased by combination treatment in both of TRAIL-resistant and -sensitive cells. These results suggest that PT sensitizes TRAIL-induced apoptosis via death receptor-independent and mitochondrial-dependent pathway. Combination treatment using PT and TRAIL might offer an effective strategy to overcome TRAIL resistance in certain CRC cells. Citation Format: Se Lim Kim, Sang-Wook Kim, Soo-Teik Lee, Seong Hun Kim, In Hee Kim, Seung Ok Lee, Dae Ghon Kim. Parthenolide sensitizes colorectal cancer cells to TRAIL-induced apoptosis by regulating mitochonrial pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2014-789

Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that has been implicated in breast tumor biology. Breast cancers commonly express PTHrP, and when such tumors are grown in bone in murine models, PTHrP results in enhanced osteolysis. Whilst clinical studies have determined that PTHrP expression by primary breast cancers was an independent predictor of improved prognosis (Henderson et al., 2006). Thus, the action of PTHrP upon breast cancers directly remains to be elucidated. The aim of this study was to assess the interaction of PTHrP with molecules associated with cell survival and cell death pathways, such as Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces apoptosis in a wide range of transformed cells but not in normal cells. To understand the role of PTHrP in relation to TRAIL in breast cancer cells, PTHrP was overexpressed in two breast cancer cell lines; MDA-MB-231 (TRAIL-sensitive) and MCF-7 (TRAIL-resistant), and functional assays were performed. Over-expression of PTHrP in both MCF-7 and MDA-MB-231 cells enhanced cell cycle progression and cell growth rate in comparison to parental and vector controls. However, treatment of PTHrP overexpressing breast cancer cells with TRAIL resulted in an enhancement of TRAIL-induced apoptosis in the TRAIL sensitive cell line, MDA-MB-231, and a sensitisation to TRAIL-induced apoptosis in the TRAIL-resistant cell line, MCF-7. Western blot showed decreased levels of Caspase-10, Caspase-9, Caspase-6, Capase-7 precursor proteins and increased PARP cleavage levels in PTHrP overexpressing cells treated with TRAIL when compared to controls. There was no change observed in levels of Caspase-8 percursor protein, suggesting that the apoptosis signal is acting via the intrinsic pathway in PTHrP overexpressing cells. To determine TRAIL sensitivity in MCF-7 cells overexpressing PTHrP, TRAIL receptor mRNA and protein expression was assessed by qRT-PCR and FACS analysis. TRAIL receptors; DR4, DR5, DcR1 and DcR2, mRNA expression was not altered in PTHrP overexpressing cells. However, flow cytometric analysis demonstrated an increase in cell surface expression of both death receptors (DR4 and DR5) in cells overexpressing PTHrP. To determine whether TRAIL-induced apoptosis in PTHrP overexpressing cells was signaling either through DR4, DR5 or a combination of both death receptors, antagonistic antibodies against DR4 or DR5 were used. Pre-incubation of PTHrP overexpressing cells with DR5 antagonistic antibody attenuated TRAIL-induced apoptosis. This demonstrated that TRAIL preferentially signals through DR5 in the PTHrP overexpressing cells to activate the intrinsic apoptotic pathway. In conclusion, these results demonstrate a new function for PTHrP in breast cancer and may provide utility when TRAIL is used as a treatment for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4886. doi:1538-7445.AM2012-4886

Expression of TNF-related apoptosis-inducing ligand (TRAIL) in megakaryocytes and platelets

Objective To tumor necrosis factor- related apoptosis- inducing ligand(TRAIL)combined with a protease inhibitor MG132 observed in human lung adenocarcinoma cell line GLC- 82 inhibition of proliferation and platelet derived growth factor receptor α(PDGFRα)- phospholipase Cγ1(PLCγ1)-protein kinase Cα(PKCα)signal transduction pathway. Methods Methyl thiazol tetrazolium(MTT)assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis, using SP immunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1,PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCαprotein expression. Results MTT assay inhibition of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell growth detection. The inhibitory rates were(34.92±3.95)%,(33.17±1.78)% and(76.96±1.28)%, respectively; using flow cytometry to detect the effects of TRAIL combined with a protease inhibitor MG132 on GLC- 82 cell apoptosis. Apoptosis rates of the four group were 6.78%, 38.72%, 45.60% and 91.74%, respectively. Using SPimmunohistochemical detection of protease inhibitors MG132 joint expression of TRAIL- associated protein PDGFRα, PLCγ1, PKCα's, Western blotting to determine the effect of protease inhibitors MG132 TRAIL combined for GLC- 82 cells PDGFRα, PLCγ1, PKCα protein expression. Conclusion TRAIL combined protease inhibitor MG132 on human lung adenocarcinoma GLC- 82 cells significantly inhibited the growth, can reduce the level of GLC- 82 cells PDGFRα- PLCγ1- PKCα each protein signaling pathway, the incidence of lung cancer and PDGFRα- PLCγ1- PKCα enhanced signal transduction pathways are closely related. Key words: Tumor necrosis factor-related apoptosis-inducing ligand; MG132; Lung adenocarcinoma; Signal transduction

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.