Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers ...including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFalpha strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFalpha treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFalpha. Although inhibition of TNFalpha with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derived TNFalpha plays a profound role in malignancy of PDAC, and inhibition of TNFalpha represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.
To study novel treatment modalities for pancreatic ductal adenocarcinoma (PDAC), we need to transfer the knowledge from in vitro to in vivo. It is important to mirror the clinical characteristics of ...the typically local invasive growth of pancreatic cancer and the distant spread resulting in liver metastasis. Notably, for xenotransplant studies using human specimen, two models, i.e. subcutaneous (s.c.) and orthotopic (o.t.) transplantation are widely used.
The subcutaneously and orthotopically inoculated Colo357 Bcl-xL cell-derived tumors were directly compared with and without TNF-related apoptosis inducing ligand (TRAIL) treatment. The size of primary tumors, number of liver metastasis and the histologic markers Ki67, M30, TNF-α and CD31 were assessed.
Upon TRAIL treatment, the primary tumors did not change their size, neither in the s.c. nor in the o.t. approaches. But when s.c. was compared to o.t., the size of the s.c. tumors was more than two-fold bigger than that of the o.t. tumors (P < 0.01). However, mice with orthotopically inoculated PDAC cells developed liver metastasis upon TRAIL treatment much more frequently (n = 13/17) than mice with subcutaneously inoculated PDAC cells (n = 1/11) (P < 0.01). As a likely driving force for this increased metastasis, a higher TNF-α staining intensity in the o.t. tumors was observed by immunohistochemistry.
These data from a direct side-by-side comparison underline the importance of the proper inoculation site of the PDAC cells. Local invasion and liver metastases are a hallmark of PDAC in the clinic; the o.t. model is clearly superior in reflecting this setting. Moreover, a serious side-effect of a possible new therapeutic compound became obvious only in the o.t. model.
Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. ...However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H
+
-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches.
Different clinical presentations and prognoses have been implied between pancreatic head and body/tail cancers. We aimed to identify the prognostic relevance of primary tumor location in patients ...undergoing resection for pancreatic ductal adenocarcinoma (PDAC). Thirty-two pairs of patients with strictly matched early stage (II) pancreatic head and body/tail cancers were enrolled. The molecular feature of the two subtypes of PDAC was assessed on the level of miRNA expression. Out of the 64 patients, 34 (53.1%) had tumor recurrence after radical resection during the follow-up period (2.3 ± 0.8 years). Both overall and tumor-free survival were significantly higher in the patients with pancreatic body/tail cancer compared with those with pancreatic head cancer. Patient age and tumor location were the independent prognostic factors for tumor recurrence. A remarkably lower expression of miR-501-3p and higher expression of miR-375 were found and were further verified in pancreatic body/tail cancer tissues compared with pancreatic head cancer tissues. The low expression of miR-501-3p was significantly associated with a low risk of tumor recurrence. Both, subcutaneous and orthotopic PDAC mouse models presented highly invasive tumor phenotypes upon up-regulated miR-501-3p expression. An in vitro study showed that miR-501-3p promoted the invasiveness of PDAC cells possibly via suppressing E-cadherin. In summary, at resectable early stage, pancreatic body/tail cancer presents a less malignant phenotype associated with deregulation of miR-501-3p compared with pancreatic head cancer.
Acquired immune evasion is one of the mechanisms that contributes to the dismal prognosis of cancer. Recently, we observed that different γδ T cell subsets as well as CD8
αβ T cells infiltrate the ...pancreatic tissue. Interestingly, the abundance of γδ T cells was reported to have a positive prognostic impact on survival of cancer patients. Since γδ T cells utilize TNF-related apoptosis inducing ligand (TRAIL) for killing of tumor cells in addition to granzyme B and perforin, we investigated the role of the TRAIL-/TRAIL-R system in γδ T cell-cytotoxicity toward pancreatic ductal adenocarcinoma (PDAC) and other cancer cells. Coculture of the different cancer cells with γδ T cells resulted in a moderate lysis of tumor cells. The lysis of PDAC Colo357 cells was independent of TRAIL as it was not inhibited by the addition of neutralizing anti-TRAIL antibodies or TRAIL-R2-Fc fusion protein. In accordance, knockdown (KD) of death receptors TRAIL-R1 or TRAIL-R2 in Colo357 cells had no effect on γδ T cell-mediated cytotoxicity. However, KD of decoy receptor TRAIL-R4, which robustly enhanced TRAIL-induced apoptosis, interestingly, almost completely abolished the γδ T cell-mediated lysis of these tumor cells. This effect was associated with a reduced secretion of granzyme B by γδ T cells and enhanced PGE2 production as a result of increased expression level of synthetase cyclooxygenase (COX)-2 by TRAIL-R4-KD cells. In contrast, knockin of TRAIL-R4 decreased COX-2 expression. Importantly, reduced release of granzyme B by γδ T cells cocultured with TRAIL-R4-KD cells was partially reverted by bispecific antibody HER2xCD3 and led in consequence to enhanced lysis of tumor cells. Likewise, inhibition of COX-1 and/or COX-2 partially enhanced γδ T cell-mediated lysis of TRAIL-R4-KD cells. The combination of bispecific antibody and COX-inhibitor completely restored the lysis of TRAIL-R4-KD cells by γδ T cells. In conclusion, we uncovered an unexpected novel role of TRAIL-R4 in tumor cells. In contrast to its known pro-tumoral, anti-apoptotic function, TRAIL-R4 augments the anti-tumoral cytotoxic activity of γδ T cells.
The death receptors TRAIL-R1 and TRAIL-R2 are frequently overexpressed in cancer and there is an emerging evidence for their important role in malignant progression, also in the case of pancreatic ...ductal adenocarcinoma (PDAC). In their canonical localization at the plasma membrane, TRAIL-R1/-R2 may induce cell death and/or pro-inflammatory signaling leading to cell migration, invasion and metastasis. Although, they have repeatedly been found intracellular, in the cytoplasm and in the nucleus, their functions in intracellular locations are still not well understood. Likewise, studies dealing with the prognostic relevance of TRAIL-Rs located in particular cellular compartments are very rare. For PDAC, the correlation of nuclear TRAIL-R2 with worse patients' prognosis has been shown recently. Corresponding data on TRAIL-R1 are not available so far.
In the present study we analyzed the expression of TRAIL-R1 in 106 PDACs and 28 adjacent, peritumoral non-malignant pancreatic ducts with special emphasis on its cytoplasmic and nuclear localization and correlated the immunohistochemical findings with clinico-pathological patient characteristics.
TRAIL-R1 was found in 93.4% of all PDAC samples. Cytoplasmic staining was present with very similar intensity in tumor and normal tissue. In contrast, nuclear TRAIL-R1 staining was significantly stronger in tumor compared to normal tissue (p = 0.006). Interestingly, we found that the number of cells with cytoplasmic TRAIL-R1 staining negatively correlates with tumor grading (p = 0.043). No such correlation could be detected for nuclear TRAIL-R1. Neither, cytoplasmic nor nuclear TRAIL-R1 staining showed a correlation with other clinico-pathological parameter such as pTNM categories. However, Kaplan-Meier analyses revealed significantly prolonged median survival of patients with positive cytoplasmic TRAIL-R1 expression in more than 80% of tumor cells compared to patients with tumors containing a smaller quantity of cells positively stained for cytoplasmic TRAIL-R1 (20 vs. 8 months; p = 0.004).
Cytoplasmic TRAIL-R1 is a positive prognostic marker for patients with PDAC. Our findings indicate that loss of cytoplasmic TRAIL-R1 results in recurrent disease with more malignant phenotype thus suggesting anti-tumor activities of cytoplasmic TRAIL-R1 in PDAC.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Purpose
Bone metastasis in breast cancer has been linked to activity of c-Src kinase, one of the extensively explored tyrosine kinases in cell biology. The impact of TNF-related apoptosis inducing ...ligand (TRAIL) and TRAIL receptors has just recently been integrated into this conception.
Methods
An osteotropic clone of MDA-MB-231 cells simulated a model for bone metastasis of triple-negative breast cancer (TNBC). The effects of Dasatinib, a clinically established inhibitor of Src kinases family and Abl were evaluated in vitro and in vivo. In vivo effects of Dasatinib treatment on the occurrence of skeletal metastases were tested in a xenograft mouse model after intra-cardiac injection of osteotropic MDA-MB-231-cells. Ex vivo analyses of the bone sections confirmed intraosseous growth of metastases and allowed determination of osteoclastic activity.
Results
Treatment of osteotropic MDA-MB-231 cells with Dasatinib inhibited proliferation rates in vitro. A shift in TRAIL-receptor expression towards an induction of oncogenic TRAIL-R2 was observed. In vivo, 15 of 30 mice received an intra-peritoneal treatment with Dasatinib. These mice showed significantly less skeletal metastases in bioluminescence scans. Moreover, a pronounced increase in bone volume was observed in the treatment group, as detected by µ-Computed Tomography. Dasatinib treatment also led to a greater increase in bone density in tibiae without metastatic affection, which was accompanied by reduced recruitment of osteoclasts.
Conclusion
Our observations support the concept of utilizing Dasatinib in targeting early-stage bone metastatic TNBC and sustaining bone health.
Pancreatic ductal adenocarcinoma, PDAC, is a type of tumor with late diagnosis, rapid progression and poor prognosis. It is mostly unresponsive to chemo- and radiation therapy due to a marked ...resistance against apoptosis induction via both the intrinsic and extrinsic pathway. Accordingly, also activation of death receptors of the TNF-receptor superfamily does not or hardly induces apoptosis in PDAC cells. The reasons for this resistance include expression of multiple inhibitory proteins that block apoptotic signaling at almost every step of the signaling cascade. Moreover and more importantly, death receptors such as CD95/Fas, TRAIL-R1/-R2 and TNF-R1 exhibit marked non-apoptotic functions after ligand binding leading to strong proinflammatory responses, which contribute to survival, proliferation, migration and invasion, causing an even more pronounced malignant phenotype. This non-apoptotic signal transduction is facilitated via distinct adapter proteins, such as TRAF2 that bind to death receptor complexes and stimulate proinflammatory signal pathways leading to activation of NF-κB and AP-1 transcription factors.
Therapeutic interventions in oncology utilizing death ligands, e.g. TRAIL, or triggering death receptors indirectly, must be carefully evaluated with respect to possible “side effects” and should be designed in a way counteracting non-apoptotic proinflammatory signaling.
The accumulation of perturbations in signalling pathways resulting in an apoptosis-insensitive phenotype is largely responsible for the desperate prognosis of patients with pancreatic ductal ...adenocarcinoma (PDAC). Accumulating evidence suggests that the death receptors TRAIL-R1 and TRAIL-R2 play important roles in PDAC biology by acting as either tumour suppressors through induction of cell death or tumour promoters through induction of pro-inflammatory signalling, invasion and metastasis. TRAIL-R2 can also associate with nuclear proteins and alter the maturation of micro RNAs (miRs). By genome-wide miR profiling and quantitative PCR analyses we now demonstrate that knockdown of TRAIL-R1 in PDAC cells decreased the level of mature miR-370 and led to an increased abundance of the type II receptor for transforming growth factor β (TGFβ). Transfection of cells with an artificial miR-370-3p decreased the levels of TGFβ-RII. We further show that transient expression of the miR-370 mimic decreased TGFβ1-induced expression of
encoding plasminogen activator-inhibitor 1 and partially relieved TGFβ1-induced growth inhibition. Moreover, stable TRAIL-R1 knockdown in Colo357 cells increased TGFβ1-induced
expression and this effect was partially reversed by transient expression of the miR-370 mimic. Finally, after transient knockdown of TRAIL-R1 in Panc1 cells there was a tendency towards enhanced activation of Smad2 and JNK1/2 signalling by exogenous TGFβ1. Taken together, our study reveals that TRAIL-R1 through regulation of miR-370 can decrease the sensitivity of PDAC cells to TGFβ and therefore represents a potential tumour suppressor in late-stage PDAC.
Binding of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to the plasma membrane TRAIL-R1/-R2 selectively kills tumor cells. This discovery led to evaluation of TRAIL-R1/-R2 as ...targets for anti-cancer therapy, yet the corresponding clinical trials were disappointing. Meanwhile, it emerged that many cancer cells are TRAIL-resistant and that TRAIL-R1/-R2-triggering may lead to tumor-promoting effects. Intriguingly, recent studies uncovered specific functions of long ignored intracellular TRAIL-R1/-R2, with tumor-promoting functions of nuclear (n)TRAIL-R2 as the regulator of let-7-maturation. As nuclear trafficking of TRAIL-Rs is not well understood, we addressed this issue in our present study. Cell surface biotinylation and tracking of biotinylated proteins in intracellular compartments revealed that nTRAIL-Rs originate from the plasma membrane. Nuclear TRAIL-Rs-trafficking is a fast process, requiring clathrin-dependent endocytosis and it is TRAIL-dependent. Immunoprecipitation and immunofluorescence approaches revealed an interaction of nTRAIL-R2 with the nucleo-cytoplasmic shuttle protein Exportin-1/CRM-1. Mutation of a putative nuclear export sequence (NES) in TRAIL-R2 or the inhibition of CRM-1 by Leptomycin-B resulted in the nuclear accumulation of TRAIL-R2. In addition, TRAIL-R1 and TRAIL-R2 constitutively localize to chromatin, which is strongly enhanced by TRAIL-treatment. Our data highlight the novel role for surface-activated TRAIL-Rs by direct trafficking and signaling into the nucleus, a previously unknown signaling principle for cell surface receptors that belong to the TNF-superfamily.
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