Natural Killer Cell Receptors and Endometriosis: A Systematic Review

Endometriosis is a chronic inflammatory disorder, characterized by the presence of endometrial cells outside the uterine cavity. An increasing number of studies correlate the immune system with endometriosis, particularly NK receptors (NKR), which have been suggested to play an essential role in the pathogenesis of the disease. This systematic review aims to enlighten the role of NKR in endometriosis. A literature search was performed independently by two reviewers, to identify studies assessing the role of NKR in endometriosis. In total, 18 studies were included. Endometriosis pathogenesis seems to be marked by the overexpression of NK inhibitor receptors (KIRS), namely, CD158a+, KIR2DL1, CD94/NKG2A, PD-1, NKB1, and EB6, and inhibiting ligands such as PD-L1, HLA-E, HLA-G, and HLA-I. Concurrently, there is a decrease in NK-activating receptors and natural cytotoxicity receptors (NCRs), such as NKp46, NKp30, and NKG2D. The immune shift from NK surveillance to NK suppression is also apparent in the greater relative number of ITIM domains compared with ITAM domains in NKRs. In conclusion, NK receptor activity seems to dictate the immunocompetency of women to clear endometriotic cells from the peritoneal cavity. Future research could explore NKRs as therapeutic targets, such as that which is now well established in cancer therapy through immunotherapy.     

Convergent Effects of Resveratrol and PYK2 on Prostate Cells

Resveratrol, a dietary polyphenol, is under consideration as chemopreventive and chemotherapeutic agent for several diseases, including cancer. However, its mechanisms of action and its effects on non-tumor cells, fundamental to understand its real efficacy as chemopreventive agent, remain largely unknown. Proline-rich tyrosine kinase 2 (PYK2), a non-receptor tyrosine kinase acting as signaling mediator of different stimuli, behaves as tumor-suppressor in prostate. Since, PYK2 and RSV share several fields of interaction, including oxidative stress, we have investigated their functional relationship in human non-transformed prostate EPN cells and in their tumor-prone counterpart EPN-PKM, expressing a PYK2 dead-kinase mutant. We show that RSV has a strong biological activity in both cell lines, decreasing ROS production, inducing morphological changes and reversible growth arrest, and activating autophagy but not apoptosis. Interestingly, the PYK2 mutant increases basal ROS and autophagy levels, and modulates the intensity of RSV effects. In particular, the anti-oxidant effect of RSV is more potent in EPN than in EPN-PKM, whereas its anti-proliferative and pro-autophagic effects are more significant in EPN-PKM. Consistently, PYK2 depletion by RNAi replicates the effects of the PKM mutant. Taken together, our results reveal that PYK2 and RSV act on common cellular pathways and suggest that RSV effects on prostate cells may depend on mutational-state or expression levels of PYK2 that emerges as a possible mediator of RSV mechanisms of action. Moreover, the observation that resveratrol effects are reversible and not associated to apoptosis in tumor-prone EPN-PKM cells suggests caution for its use in humans.     

PEBP4 Directs the Malignant Behavior of Hepatocellular Carcinoma Cells via Regulating mTORC1 and mTORC2

Phosphatidylethanolamine binding protein 4 (PEBP4) is an understudied multifunctional small protein. Previous studies have shown that the expression of PEBP4 is increased in many cancer specimens, which correlates to cancer progression. The present study explored the mechanism by which PEBP4 regulates the growth and progression of hepatocellular carcinoma cells. Thus, we showed that knockdown of PEBP4 in MHCC97H cells, where its expression was relatively high, diminished activities of serine/threonine protein kinase B (PKB, also known as Akt), mammalian target of rapamycin complex 1(mTORC1), and mTORC2, events that were not restored by insulin-like growth factor 1 (IGF-1). Conversely, overexpression of PEBP4 in MHCC97L cells with the low endogenous level yielded opposite effects. Furthermore, physical association of PEBP4 with Akt, mTORC1, and mTORC2 was observed. Interestingly, introduction of AktS473D mutant, bypassing phosphorylation by mTORC2, rescued mTORC1 activity, but without effects on mTORC2 signaling. In contrast, the effect of PEBP4 overexpression on the activity of mTORC1 but not that of mTORC2 was suppressed by MK2206, a specific inhibitor of Akt. In conjunction, PEBP4 knockdown-engendered reduction of cell proliferation, migration and invasion was partially rescued by Akt S473D while increases in these parameters induced by overexpression of PEBP4 were completely abolished by MK2206, although the expression of epithelial mesenchymal transition (EMT) markers appeared to be fully regulated by the active mutant of Akt. Finally, knockdown of PEBP4 diminished the growth of tumor and metastasis, whereas they were enhanced by overexpression of PEBP4. Altogether, our study suggests that increased expression of PEBP4 exacerbates malignant behaviors of hepatocellular cancer cells through cooperative participation of mTORC1 and mTORC2.     

A Hot Topic: Cancer Immunotherapy and Natural Killer Cells

Despite significant progress in recent years, the therapeutic approach of the multiple different forms of human cancer often remains a challenge. Besides the well-established cancer surgery, radiotherapy and chemotherapy, immunotherapeutic strategies gain more and more attention, and some of them have already been successfully introduced into the clinic. Among these, immunotherapy based on natural killer (NK) cells is considered as one of the most promising options. In the present review, we will expose the different possibilities NK cells offer in this context, compare data about the theoretical background and mechanism(s) of action, report some results of clinical trials and identify several very recent trends. The pharmaceutical industry is quite interested in NK cell immunotherapy, which will benefit the speed of progress in the field.     

Visfatin Mediates SCLC Cells Migration across Brain Endothelial Cells through Upregulation of CCL2

Small-cell lung cancer (SCLC) is characterized as an aggressive tumor with brain metastasis. Although preventing SCLC metastasis to the brain is immensely important for survival, the molecular mechanisms of SCLC cells penetrating the blood–brain barrier (BBB) are largely unknown. Recently, visfatin has been considered as a novel pro-inflammatory adipocytokine involved in various cancers. Herein, we present evidence that elevated levels of visfatin in the serum of SCLC patients were associated with brain metastasis, and visfain was increased in NCI-H446 cells, a SCLC cell line, during interacting with human brain microvascular endothelial cells (HBMEC). Using in vitro BBB model, we found that visfatin could promote NCI-H446 cells migration across HBMEC monolayer, while the effect was inhibited by knockdown of visfatin. Furthermore, our findings indicated that CC chemokine ligand 2 (CCL2) was involved in visfatin-mediated NCI-H446 cells transendothelial migtation. Results also showed that the upregulation of CCL2 in the co-culture system was reversed by blockade of visfatin. In particular, visfatin-induced CCL2 was attenuated by specific inhibitor of PI3K/Akt signaling in NCI-H446 cells. Taken together, we demonstrated that visfatin was a prospective target for SCLC metastasis to brain, and understanding the molecular mediators would lead to effective strategies for inhibition of SCLC brain metastasis.     

Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.     

Synergistic Effects of Venetoclax and Daratumumab on Antibody-Dependent Cell-Mediated Natural Killer Cytotoxicity in Multiple Myeloma

The prognosis of multiple myeloma (MM) has drastically improved owing to the development of new drugs, such as proteasome inhibitors and immunomodulatory drugs. Nevertheless, MM is an extremely challenging disease, and many patients are still refractory to the existing therapies, thus requiring new treatment alternatives. Venetoclax is a selective, orally bioavailable inhibitor of BCL-2 that shows efficacy in MM not only as a single agent but also in combination therapy, especially for MM patients with translocation t(11;14). However, many patients are refractory to this drug. Here, we treated the MM cell lines KMS12PE and KMS27 with a combination treatment of venetoclax targeting BCL-2 and daratumumab targeting CD38 to evaluate the synergistic cytotoxicity of these drugs in vitro. MM cell lines were co-cultured with natural killer (NK) cells at an effector:target ratio of 0.3:1 in the presence of serial concentrations of daratumumab and venetoclax, and the resulting apoptotic MM cells were detected by flow cytometry using annexin V. These results indicated that the antibody-dependent cell-mediated NK cytotoxicity was enhanced in KMS12PE and KMS27 cells harboring t(11;14) with a high BCL-2 expression, suggesting that the combination treatment of venetoclax and daratumumab should be especially effective in patients with these characteristics.     

Resveratrol Protects against 2-Bromopropane-Induced Apoptosis and Disruption of Embryonic Development in Blastocysts

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. In the present work, we show that 2-BP induces apoptosis in the inner cell mass of mouse blastocysts, and inhibits cell proliferation. Both effects are suppressed by resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties. 2-BP-treated blastocysts displayed lower levels of implantation (compared to controls) when plated on culture dishes in vitro, and a reduced ability to proceed to later stages of embryonic development. Pretreatment with resveratrol prevented 2-BP-induced disruption of embryonic development, both in vitro and in vivo. Further investigation of these processes revealed that 2-BP directly promotes ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-3, whereas resveratrol effectively blocks 2-BP-induced ROS production and the accompanying apoptotic biochemical changes. Our results collectively imply that 2-BP triggers the mitochondrion-dependent apoptotic pathway via ROS generation, and the antioxidant activity of resveratrol prevents 2-BP-induced toxicity.     

Separation and Identification of Resveratrol Butyrate Ester Complexes and Their Bioactivity in HepG2 Cell Models

Resveratrol butyrate ester (RBE) complexes have demonstrated higher antioxidant capacity and anti-fat accumulation activity in previous studies. In this study, silica gel, high-performance liquid chromatography, and 1H nuclear magnetic resonance were used for separation and identification of RBE complex components. With the exception of resveratrol, five different structures of ester derivatives were separated from silica gel: 3,4′-di-O-butanoylresveratrol (ED2, 18.8%), 3-O-butanoylresveratrol (ED4, 35.7%), 4′-O-butanoylresveratrol (ED5, 4.4%), 3,5,4′-tri-O-butanoylresveratrol (ED6, 1.5%), and 3,5-di-O-butanoylresveratrol (ED7, 0.7%). Among the ester derivatives obtained, ED2 and ED4 were the main ester derivatives in the RBE complex. Thus, the cellular antioxidant activities of the RBE mixture, ED2, and ED4 were evaluated. Results showed that the antioxidant capacity of ED2 and ED4 was higher than that of the RBE mixture, demonstrating that the number and position of butyrate esterification sites are related to cell survival rate and antioxidant capacity. This study is the first to report the successful isolation, structural identification, and cellular biological antioxidant activity of RBE complex derivatives, which are key characteristics for the potential practical application of RBE complexes.     

抗体阻断表面黏附分子DNAM-1和LFA-1对人自然杀伤细胞肿瘤杀伤活性的抑制作用

目的探讨抗体阻断表面黏附分子DNAM-1和LFA-1对体外扩增的人自然杀伤(Natural killer,NK)细胞的肿瘤杀伤活性的影响。方法体外活化人外周血单个核细胞来源的NK细胞,并检测其表面黏附分子DNAM-1和LFA-1的表达水平;通过Trans-well阻隔试验,阻断NK细胞与靶细胞接触,考察细胞-细胞直接接触对NK细胞杀伤活性的影响;通过NK细胞毒性试验,引入抗DNAM-1单克隆抗体和抗LFA-1单克隆抗体,阻断相应信号途径,考察相关黏附分子在NK细胞肿瘤杀伤过程中的作用。结果体外活化的NK细胞高表达表面黏附分子DNAM-1和LFA-1。阻断NK细胞与靶细胞接触,NK细胞的肿瘤杀伤效应大大降低。应用单克隆抗体阻断DNAM-1或LFA-1信号途径,可显著降低NK细胞肿瘤的杀伤效应;联合阻断DNAM-1和LFA-1信号途径,可进一步降低NK的细胞杀伤效应。结论细胞-细胞间直接接触是NK细胞发挥肿瘤杀伤效应的重要机制,表面黏附分子DNAM-1和LFA-1介导的信号途径对维持NK细胞肿瘤杀伤活性具有重要意义。     

Ligation of TLR Homologue CD180 of B Cells Activates the PI3K/Akt/mTOR Pathway in Systemic Sclerosis and Induces a Pathological Shift in the Expression of BAFF Receptors

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) pathways are known to play a key role in B-cell activation and fibrosis in systemic sclerosis (SSc). Receptors of B-cell activator factor (BAFF) utilize these pathways, which can be influenced by Toll-like receptors (TLRs), as TLRs can alter the expression of BAFF-binding receptors. Our results show that B-cell stimulation via TLR homologue CD180 phosphorylates Akt in diffuse cutaneous SSc (dcSSc) to a lower extent than in healthy controls (HCs). We found basal downregulated BAFF receptor (BAFF-R) and enhanced transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) expression in dcSSc B cells, which might enhance the formation of autoantibody-secreting plasma cells. Moreover, this pathological shift was observed in naive B cells, emphasizing the importance of their increase in SSc. Additionally, we measured higher serum levels of autoantibodies to BAFF in dcSSc patients, suggesting that an imbalance in the complex system of BAFF/anti-BAFF autoantibodies/BAFF-binding receptors may contribute to the development of SSc. Anti-CD180 antibody treatment had opposite effects on the expression of BAFF-R and TACI in HC B cells, resulting in similar levels as observed in SSc B cells without stimulation, which argues against the usefulness of such therapy in SSc.     

The mTORC2 Regulator Homer1 Modulates Protein Levels and Sub-Cellular Localization of the CaSR in Osteoblast-Lineage Cells

We recently found that, in human osteoblasts, Homer1 complexes to Calcium-sensing receptor (CaSR) and mediates AKT initiation via mechanistic target of rapamycin complex (mTOR) complex 2 (mTORC2) leading to beneficial effects in osteoblasts including β-catenin stabilization and mTOR complex 1 (mTORC1) activation. Herein we further investigated the relationship between Homer1 and CaSR and demonstrate a link between the protein levels of CaSR and Homer1 in human osteoblasts in primary culture. Thus, when siRNA was used to suppress the CaSR, we observed upregulated Homer1 levels, and when siRNA was used to suppress Homer1 we observed downregulated CaSR protein levels using immunofluorescence staining of cultured osteoblasts as well as Western blot analyses of cell protein extracts. This finding was confirmed in vivo as the bone cells from osteoblast specific CaSR−/− mice showed increased Homer1 expression compared to wild-type (wt). CaSR and Homer1 protein were both expressed in osteocytes embedded in the long bones of wt mice, and immunofluorescent studies of these cells revealed that Homer1 protein sub-cellular localization was markedly altered in the osteocytes of CaSR−/− mice compared to wt. The study identifies additional roles for Homer1 in the control of the protein level and subcellular localization of CaSR in cells of the osteoblast lineage, in addition to its established role of mTORC2 activation downstream of the receptor.     

Construction of IL-13 Receptor α2-Targeting Resveratrol Nanoparticles against Glioblastoma Cells: Therapeutic Efficacy and Molecular Effects

Glioblastoma multiforme (GBM) is the most common lethal primary brain malignancy without reliable therapeutic drugs. IL-13Rα2 is frequently expressed in GBMs as a molecular marker. Resveratrol (Res) effectively inhibits GBM cell growth but has not been applied in vivo because of its low brain bioavailability when administered systemically. A sustained-release and GBM-targeting resveratrol form may overcome this therapeutic dilemma. To achieve this goal, encapsulated Res 30 ± 4.8 nm IL-13Rα2-targeting nanoparticles (Pep-PP@Res) were constructed. Ultraviolet spectrophotometry revealed prolonged Res release (about 25%) from Pep-PP@Res in 48 h and fluorescent confocal microscopy showed the prolonged intracellular Res retention time of Pep-PP@Res (>24 h) in comparison with that of free Res (<4 h) and PP@Res (<4 h). MTT and EdU cell proliferation assays showed stronger suppressive effects of Pep-PP@Res on rat C6 GBM cells than that of PP@Res (p = 0.024) and Res (p = 0.009) when used twice for 4 h/day. Pep-PP@Res had little toxic effect on normal rat brain cells. The in vivo anti-glioblastoma effects of Res can be distinctly improved in the form of Pep-PP@Res nanoparticles via activating JNK signaling, upregulating proapoptosis gene expression and, finally, resulting in extensive apoptosis. Pep-PP@Res with sustained release and GBM-targeting properties would be suitable for in vivo management of GBMs.     

Primary TSC2-/meth Cells Induce Follicular Neogenesis in an Innovative TSC Mouse Model

Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient’s life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2^-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2^-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2^-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.     

Resveratrol Treatment Prevents Increase of Mast Cells in Both Murine OVA Enteritis and IL-10−/− Colitis

Mast cells are involved in allergic and other inflammatory diseases. The polyphenol resveratrol is known for its anti-inflammatory properties and may be used as nutraceutical in mast cell associated diseases. We analyzed the effect of resveratrol on mast cells in vivo in ovalbumin-induced allergic enteritis as well as experimental colitis in IL-10^−/− mice which received resveratrol via drinking water. Treatment with resveratrol prevented the increase in mast cells in both allergic enteritis and chronic colitis in duodenum as well as in colon. Further, it delayed the onset of diseases symptoms and ameliorated diseases associated parameters such as tissue damage as well as inflammatory cell infiltration in affected colon sections. In addition to the findings in vivo, resveratrol inhibited IgE-dependent degranulation and expression of pro-inflammatory cytokines such as TNF-α in IgE/DNP-activated as well as in LPS-activated bone marrow-derived mast cells. These results indicate that resveratrol may be considered as an anti-allergic and anti-inflammatory plant-derived component for the prevention or treatment of mast cell-associated disorders of the gastrointestinal tract.     

Deciphering the Immunological Phenomenon of Adaptive Natural Killer (NK) Cells and Cytomegalovirus (CMV)

Natural killer (NK) cells play a significant and vital role in the first line of defense against infection through their ability to target cells without prior sensitization. They also contribute significantly to the activation and recruitment of both innate and adaptive immune cells through the production of a range of cytokines and chemokines. In the context of cytomegalovirus (CMV) infection, NK cells and CMV have co-evolved side by side to employ several mechanisms to evade one another. However, during this co-evolution the discovery of a subset of long-lived NK cells with enhanced effector potential, increased antibody-dependent responses and the potential to mediate immune memory has revolutionized the field of NK cell biology. The ability of a virus to imprint on the NK cell receptor repertoire resulting in the expansion of diverse, highly functional NK cells to this day remains a significant immunological phenomenon that only occurs in the context of CMV. Here we review our current understanding of the development of these NK cells, commonly referred to as adaptive NK cells and their current role in transplantation, infection, vaccination and cancer immunotherapy to decipher the complex role of CMV in dictating NK cell functional fate.     

Evidence That β1-Integrin Is Required for the Anti-Viability and Anti-Proliferative Effect of Resveratrol in CRC Cells

The β1-integrin receptor is broadly expressed on tumor and other cells in the tumor microenvironment (TME), and is an unfavorable prognostic factor for cancers. Nature-derived resveratrol has preventive and apoptotic effects on tumors, but whether resveratrol can exert its suppressive actions on TME-induced tumorigenesis through β1-integrin on the surface of CRC cells is still unknown. HCT116 or SW480 cells were exposed to inhibitory antibodies against β1-integrin, bacitracin (selective β1-integrin inhibitor), integrin-binding RGD (Arg-Gly-Asp) peptide, and/or resveratrol. We evaluated the anti-tumor actions and signaling impacts of resveratrol in colorectal cancer (CRC)-TME. We found that resveratrol completely altered the β1-integrin distribution pattern and expression on the surface of CRC cells in TME. Moreover, resveratrol down-regulated CRC cell proliferation, colony formation, viability, and up-regulated apoptosis in a concentration-dependent way. These actions of resveratrol were antagonized mainly by inhibitory antibodies against β1-integrin but not β5-integrin, and by an integrin-binding RGD peptide but not by RGE peptide, and by bacitracin in TME. Similarly, resveratrol-blocked TME-induced p65-NF-kB and its promoted gene markers linked to proliferation (cyclin D1), invasion (focal adhesion kinase, FAK), or apoptosis (caspase-3), were largely abrogated by anti-β1-integrin or RGD peptide, suggesting that β1-integrin is a potential transmission pathway for resveratrol/integrin down-stream signaling in CRC cells. The current results highlight, for the first time, the important gateway role of β1-integrins as signal carriers for resveratrol on the surfaces of HCT116 and SW480 cells, and their functional cooperation for the modulatory effects of resveratrol on TME-promoted tumorigenesis.     

Evodiamine Induces Apoptosis and Enhances TRAIL-Induced Apoptosis in Human Bladder Cancer Cells through mTOR/S6K1-Mediated Downregulation of Mcl-1

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anti-cancer agents, has been considered as a new strategy for anti-cancer therapy. In this study, we demonstrated that evodiamine, a quinolone alkaloid isolated from the fruit of Evodia fructus, induced apoptosis and enhanced TRAIL-induced apoptosis in human bladder cancer cells. To elucidate the underlying mechanism, we found that evodiamine significantly reduced the protein levels of Mcl-1 in 253J and T24 bladder cancer cells, and overexpression of this molecule attenuated the apoptosis induced by evodiamine alone, or in combination with TRAIL. Further experiments revealed that evodiamine did not affect the mRNA level, proteasomal degradation and protein stability of Mcl-1. On the other hand, evodiamine inhibited the mTOR/S6K1 pathway, which usually regulates protein translation; moreover, knockdown of S6K1 with small interfering RNA (siRNA) effectively reduced Mcl-1 levels, indicating evodiamine downregulates c-FLIP through inhibition of mTOR/S6K1 pathway. Taken together, our results indicate that evodiamine induces apoptosis and enhances TRAIL-induced apoptosis possibly through mTOR/S6K1-mediated downregulation of Mcl-1; furthermore, these findings provide a rationale for the combined application of evodiamine with TRAIL in the treatment of bladder cancer.