Due to their ability to preferentially induce cell death in tumor cells, while sparing healthy cells, TNF-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL-R1 or ...anti-TRAIL-R2-specific antibodies are under clinical investigations for cancer-treatment. However, TRAIL-Rs may also induce signaling pathways, which result in malignant progression. TRAIL receptors are transcriptionally upregulated via wild-type p53 following radio- or chemotherapy. Nevertheless, the impact of p53 status on the expression and signaling of TRAIL-Rs is not fully understood. Therefore, we analyzed side by side apoptotic and non-apoptotic signaling induced by TRAIL or the agonistic TRAIL-R-specific antibodies Mapatumumab (anti-TRAIL-R1) and Lexatumumab (anti-TRAIL-R2) in the two isogenic colon carcinoma cell lines HCT116 p53+/+ and p53-/-. We found that HCT116 p53+/+ cells were significantly more sensitive to TRAIL-R-triggering than p53-/- cells. Similarly, A549 lung cancer cells expressing wild-type p53 were more sensitive to TRAIL-R-mediated cell death than their derivatives with knockdown of p53. Our data demonstrate that the contribution of p53 in regulating TRAIL-R-induced apoptosis does not correlate to the levels of TRAIL-Rs at the plasma membrane, but rather to p53-mediated upregulation of Bax, favouring the mitochondrial amplification loop. Consistently, stronger caspase-9 and caspase-3 activation as well as PARP-cleavage was observed following TRAIL-R-triggering in HCT116 p53+/+ compared to HCT116 p53-/- cells. Interestingly, HCT116 p53+/+ cells showed also a more potent activation of non-canonical TRAIL-R-induced signal transduction pathways like JNK, p38 and ERK1/ERK2 than p53-/- cells. Likewise, these cells induced IL-8 expression in response to TRAIL, Mapatumumab or Lexatumumab significantly stronger than p53-/- cells. We obtained similar results in A549 cells with or without p53-knockdown and in the two isogenic colon cancer cell lines RKO p53+/+ and p53-/-. In both cellular systems, we could clearly demonstrate the potentiating effects of p53 on TRAIL-R-mediated IL-8 induction. In conclusion, we found that wild-type p53 increases TRAIL-R-mediated apoptosis but simultaneously augments non-apoptotic signaling.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Green tea catechins are considered as possible cancer preventive agents for several cancer types but little is known regarding their effects on pancreatic cancer cells. The best studied catechin and ...the major polyphenol present in green tea is epigallocatechin gallate (EGCG). In the present study, we investigated the in vitro anti‐tumoral properties of EGCG on human pancreatic ductal adenocarcinoma (PDAC) cells PancTu‐I, Panc1, Panc89 and BxPC3 in comparison with the effects of two minor components of green tea catechins, catechin gallate (CG) and epicatechin gallate (ECG). We found that all three catechins inhibited proliferation of PDAC cells in a dose‐ and time‐dependent manner. Interestingly, CG and ECG exerted much stronger anti‐proliferative effects than EGCG. Western blot analyses performed with PancTu‐I cells revealed catechin‐mediated modulation of cell cycle regulatory proteins (cyclins, cyclin‐dependent kinases CDK, CDK inhibitors). Again, these effects were clearly more pronounced in CG or ECG than in EGCG‐treated cells. Importantly, catechins, in particular ECG, inhibited TNFα‐induced activation of NF‐κB and consequently secretion of pro‐inflammatory and invasion promoting proteins like IL‐8 and uPA. Overall, our data show that green tea catechins ECG and CG exhibit potent and much stronger anti‐proliferative and anti‐inflammatory activities on PDAC cells than the most studied catechin EGCG. (Cancer Sci 2011; 102: 728–734)
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important mediator of tumor immune surveillance. In addition, its potential to kill cancer cells without harming healthy cells ...led to the development of TRAIL receptor agonists, which however did not show the desired effects in clinical trials. This is caused mainly by apoptosis resistance mechanisms operating in primary cancer cells. Meanwhile, it has been realized that in addition to cell death, TRAIL also induces non-apoptotic pro-inflammatory pathways that may enhance tumor malignancy. Due to its late detection and resistance to current therapeutic options, pancreatic ductal adenocarcinoma (PDAC) is still one of the deadliest types of cancer worldwide. A dysregulated pH microenvironment contributes to PDAC development, in which the cancer cells become highly dependent on to maintain their metabolism. The impact of extracellular pH (pH
) on TRAIL-induced signaling in PDAC cells is poorly understood so far. To close this gap, we analyzed the effects of acidic and alkaline pH
, both in short-term and long-term settings, on apoptotic and non-apoptotic TRAIL-induced signaling. We found that acidic and alkaline pH
differentially impact TRAIL-induced responses, and in addition, the duration of the pH
exposition also represents an important parameter. Thus, adaptation to acidic pH
increases TRAIL sensitivity in two different PDAC cell lines, Colo357 and Panc1, one already TRAIL-sensitive and the other TRAIL-resistant, respectively. However, the latter became highly TRAIL-sensitive only by concomitant inhibition of Bcl-xL. None of these effects was observed under other pH
conditions studied. Both TRAIL-induced non-apoptotic signaling pathways, as well as constitutively expressed anti-apoptotic proteins, were regulated by acidic pH
. Whereas the non-apoptotic pathways were differently affected in Colo357 than in Panc1 cells, the impact on the anti-apoptotic protein levels was similar in both cell lines. In Panc1 cells, adaptation to either acidic or alkaline pH
blocked the activation of the most of TRAIL-induced non-apoptotic pathways. Interestingly, under these conditions, significant downregulation of the plasma membrane levels of TRAIL-R1 and TRAIL-R2 was observed. Summing up, extracellular pH influences PDAC cells' response to TRAIL with acidic pH
adaptation, showing the ability to strongly increase TRAIL sensitivity and in addition to inhibit TRAIL-induced pro-inflammatory signaling.
TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting ...pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.
In the late 1990s, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF-family, started receiving much attention for its potential in cancer therapy, due to its ...capacity to induce apoptosis selectively in tumour cells in vivo. TRAIL binds to its membrane-bound death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) inducing the formation of a death-inducing signalling complex (DISC) thereby activating the apoptotic cascade. The ability of TRAIL to also induce apoptosis independently of p53 makes TRAIL a promising anticancer agent, especially in p53-mutated tumour entities. Thus, several so-called TRAIL receptor agonists (TRAs) were developed. Unfortunately, clinical testing of these TRAs did not reveal any significant anticancer activity, presumably due to inherent or acquired TRAIL resistance of most primary tumour cells. Since the potential power of TRAIL-based therapies still lies in TRAIL's explicit cancer cell-selectivity, a desirable approach going forward for TRAIL-based cancer therapy is the identification of substances that sensitise tumour cells for TRAIL-induced apoptosis while sparing normal cells. Numerous of such TRAIL-sensitising strategies have been identified within the last decades. However, many of these approaches have not been verified in animal models, and therefore potential toxicity of these approaches has not been taken into consideration. Here, we critically summarise and discuss the status quo of TRAIL signalling in cancer cells and strategies to force tumour cells into undergoing apoptosis triggered by TRAIL as a cancer therapeutic approach. Moreover, we provide an overview and outlook on innovative and promising future TRAIL-based therapeutic strategies.
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer ...cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
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•mTRAIL-R promotes KRAS-driven lung and pancreatic cancer growth and metastasis•Human TRAIL-R2 promotes tumor growth, migration, invasion, and metastasis•Endogenous mTRAIL-R constitutively activates Rac1 in vivo in tumors•TRAIL-R2 expression positively correlates with the onset of metastasis in patients
von Karstedt et al. show that mouse TRAIL-R and human TRAIL-R2, but not TRAIL-R1, are important for the progression, invasion, and metastasis of KRAS-mutant tumors through the regulation of Rac-1.
The human pancreatic cancer cell line A818-6 can be grown in vitro either as a highly malignant, undifferentiated monolayer (ML) or as three-dimensional (3D) single layer hollow spheres (HS) ...simulating a benign, highly differentiated, duct-like pancreatic epithelial structure. This characteristic allowing A818-6 cells to switch from one phenotype to another makes these cells a unique system to characterize the cellular and molecular modifications during differentiation on one hand and malignant transformation on the other hand. Ion channels and transport proteins (transportome) have been implicated in malignant transformation. Therefore, the current study aimed to analyse the transportome gene expression profile in the A818-6 cells growing as a monolayer or as hollow spheres.
The study identified the differentially expressed transportome genes in both cellular states of A818-6 using Agilent and Nanostring arrays and some targets were validated via immunoblotting. Additionally, these results were compared to a tissue Affymetrix microarray analysis of pancreatic adenocarcinoma patients' tissues. The overall transcriptional profile of the ML and HS cells confirmed the formerly described mesenchymal features of ML and epithelial nature of HS which was further verified via high expression of E-cadherin and low expression of vimentin found in HS in comparison to ML. Among the predicted features between HS and ML was the involvement of miRNA-9 in this switch. Importantly, the bioinformatics analysis also revealed substantial number (n = 126) of altered transportome genes. Interestingly, three genes upregulated in PDAC tissue samples (GJB2, GJB5 and SLC38A6) were found to be also upregulated in ML and 3 down-regulated transportome genes (KCNQ1, TRPV6 and SLC4A) were also reduced in ML.
This reversible HS/ML in vitro system might help in understanding the pathophysiological impact of the transportome in the dedifferentiation process in pancreatic carcinogenesis. Furthermore, the HS/ML model represents a novel system for studying the role of the transportome during the switch from a more benign, differentiated (HS) to a highly malignant, undifferentiated (ML) phenotype.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background & Aims Tumor necrosis factor-related apoptosis inducing ligand (TRAIL-R1) (TNFRSF10A) and TRAIL-R2 (TNFRSF10B) on the plasma membrane bind ligands that activate apoptotic and other ...signaling pathways. Cancer cells also might have TRAIL-R2 in the cytoplasm or nucleus, although little is known about its activities in these locations. We investigated the functions of nuclear TRAIL-R2 in cancer cell lines. Methods Proteins that interact with TRAIL-R2 initially were identified in pancreatic cancer cells by immunoprecipitation, mass spectrometry, and immunofluorescence analyses. Findings were validated in colon, renal, lung, and breast cancer cells. Functions of TRAIL-R2 were determined from small interfering RNA knockdown, real-time polymerase chain reaction, Drosha-activity, microRNA array, proliferation, differentiation, and immunoblot experiments. We assessed the effects of TRAIL-R2 overexpression or knockdown in human pancreatic ductal adenocarcinoma (PDAC) cells and their ability to form tumors in mice. We also analyzed levels of TRAIL-R2 in sections of PDACs and non-neoplastic peritumoral ducts from patients. Results TRAIL-R2 was found to interact with the core microprocessor components Drosha and DGCR8 and the associated regulatory proteins p68, hnRNPA1, NF45, and NF90 in nuclei of PDAC and other tumor cells. Knockdown of TRAIL-R2 increased Drosha-mediated processing of the let-7 microRNA precursor primary let-7 (resulting in increased levels of mature let-7), reduced levels of the let-7 targets (LIN28B and HMGA2), and inhibited cell proliferation. PDAC tissues from patients had higher levels of nuclear TRAIL-R2 than non-neoplastic pancreatic tissue, which correlated with increased nuclear levels of HMGA2 and poor outcomes. Knockdown of TRAIL-R2 in PDAC cells slowed their growth as orthotopic tumors in mice. Reduced nuclear levels of TRAIL-R2 in cultured pancreatic epithelial cells promoted their differentiation. Conclusions Nuclear TRAIL-R2 inhibits maturation of the microRNA let-7 in pancreatic cancer cell lines and increases their proliferation. Pancreatic tumor samples have increased levels of nuclear TRAIL-R2, which correlate with poor outcome of patients. These findings indicate that in the nucleus, death receptors can function as tumor promoters and might be therapeutic targets.
Purpose
HMGA2 has frequently been found in benign as well as malignant tumors and a significant association between HMGA2 overexpression and poor survival in different malignancies was described. In ...pancreatic ductal adenocarcinoma (PDAC), nuclear HMGA2 expression is associated with tumor dedifferentiation and presence of lymph node metastasis. Nevertheless, the impact of HMGA2 occurrence in other cell compartments is unknown.
Methods
Intracellular distribution of HMGA2 was analyzed in PDAC (
n
= 106) and peritumoral, non-malignant ducts (
n
= 28) by immunohistochemistry. Findings were correlated with clinico-pathological data. Additionally, intracellular HMGA2 presence was studied by Western blotting of cytoplasmic and nuclear fractions of cultured cells.
Results
HMGA2 was found in the cytoplasm and in the nucleus of cultured cells. In human tumor tissue, HMGA2 was also frequently found in the cytoplasm and the nucleus of tumor cells, however, nuclear staining was generally stronger. Direct comparison from tumor tissue with corresponding non-neoplastic peritumoral tissue revealed significantly stronger expression in tumors (
p
= 0.003). Of note, the nuclear staining was significantly stronger in lymph node metastatic cell nuclei compared to primary tumor cell nuclei (
p
= 0.049). Interestingly, cytoplasmic staining positively correlated with lymph vessel (
p
= 0.004) and venous invasion (
p
= 0.046).
Conclusion
HMGA2 is a prognostic marker in PDAC. Firstly, we found a positive correlation for cytoplasmic HMGA2 expression with lympho-vascular invasion and, secondly, we found a significantly stronger nuclear expression of HMGA2 in cancer-positive lymph node nuclei compared to primary tumor cell nuclei. So far, the role of cytoplasmic HMGA2 is nearly unknown, however, our data lend support to the hypothesis that cytoplasmic HMGA2 expression is involved in nodal spread.
Despite recent advances in oncology, diagnosis, and therapy, treatment of pancreatic ductal adenocarcinoma (PDAC) is still exceedingly challenging. PDAC remains the fourth leading cause of ...cancer-related deaths worldwide. Poor prognosis is due to the aggressive growth behavior with early invasion and distant metastasis, chemoresistance, and a current lack of adequate screening methods for early detection. Consequently, novel therapeutic approaches are urgently needed. Many hopes for cancer treatment have been placed in the death ligand tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) since it was reported to induce apoptosis selectively in tumor cells in vitro and in vivo. TRAIL triggers apoptosis through binding of the trans-membrane death receptors TRAIL receptor 1 (TRAIL-R1) also death receptor 4 (DR4) and TRAIL receptor 2 (TRAIL-R2) also death receptor 5 (DR5) thereby inducing the formation of the death-inducing signaling complex (DISC) and activation of the apoptotic cascade. Unlike chemotherapeutics, TRAIL was shown to be able to induce apoptosis in a p53-independent manner, making TRAIL a promising anticancer approach for p53-mutated tumors. These cancer-selective traits of TRAIL led to the development of TRAIL-R agonists, categorized into either recombinant variants of TRAIL or agonistic antibodies against TRAIL-R1 or TRAIL-R2. However, clinical trials making use of these agonists in various tumor entities including pancreatic cancer were disappointing so far. This is thought to be caused by TRAIL resistance of numerous primary tumor cells, an insufficient agonistic activity of the drug candidates tested, and a lack of suitable biomarkers for patient stratification. Nevertheless, recently gained knowledge on the biology of the TRAIL-TRAIL-R system might now provide the chance to overcome intrinsic or acquired resistance against TRAIL and TRAIL-R agonists. In this review, we summarize the status quo of clinical studies involving TRAIL-R agonists for the treatment of pancreatic cancer and critically discuss the suitability of utilizing the TRAIL-TRAIL-R system for successful treatment.
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