Niels Smakman

Oncogenic K-Ras Turns Death Receptors Into Metastasis-Promoting Receptors in Human and Mouse Colorectal Cancer Cells

BACKGROUND & AIMSnDeath receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential.nnnMETHODSnIsogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach.nnnRESULTSnCD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis.nnnCONCLUSIONSnOncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.

Early Laparoscopic Cholecystectomy Improves Outcomes After Endoscopic Sphincterotomy for Choledochocystolithiasis

BACKGROUND & AIMSnPatients with choledochocystolithiasis generally undergo endoscopic sphincterotomy (ES) followed by laparoscopic cholecystectomy (LC). However, many patients receive this surgery 6-8 weeks after ES. There is a high conversion rate of elective LC after ES, and patients can develop recurrent biliary events during the waiting period. We investigated whether the timing of surgery influences outcome.nnnMETHODSnWe performed a randomized trial of patients with choledochocystolithiasis who underwent successful ES. Patients were randomly assigned to groups that received early LC (within 72 hours after ES, n = 49) or delayed LC (after 6-8 weeks, n = 47), based on an expected difference in conversion rate of 25% vs 5%, respectively. Conversion rate, biliary events during follow-up, duration and difficulty of surgeries, postoperative morbidity, and hospital stay were scored. Intention-to-treat analyses were performed.nnnRESULTSnGroups were comparable in age, sex, and comorbidity. There was no difference between groups in conversion rate (4.3% in early vs 8.7% in delayed group) nor were there differences in operating times and/or difficulties or hospital stays. During the waiting period for LC, 17 patients in the delayed group (36.2%) developed recurrent biliary events compared with 1 patient in the early group (P < .001).nnnCONCLUSIONSnIn a randomized trial to evaluate timing of LC after ES, recurrent biliary events occurred in 36.2% of patients whose LC was delayed for 6-8 weeks. Early LC (within 72 hours) appears to be safe and might prevent the majority of biliary events in this period following sphincterotomy.

KRASD13 Promotes Apoptosis of Human Colorectal Tumor Cells by ReovirusT3D and Oxaliplatin but not by Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand

Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRAS(D13). However, deletion of KRAS(D13) or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of beta-catenin(DeltaS45) had no effect. Likewise, KRAS(D13)- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRAS(D13) increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication.

Immunosuppression promotes reovirus therapy of colorectal liver metastases

Mortality due to colorectal cancer (CRC) is high and is associated with the development of liver metastases. Approximately 40% of human CRCs harbor an activating mutation in the KRAS oncogene. Tumor cells with activated KRAS are particularly sensitive to Reovirus T3D, a non-pathogenic oncolytic virus. The efficacy of virus-based therapies may be positively or negatively modulated by the host immune system. This study was designed to assess the effect of immunosuppression on Reovirus T3D oncolysis of established colorectal micrometastases in the liver. Mouse C26 CRC cells harbor a mutant Kras gene and are susceptible to Kras-dependent oncolysis by Reovirus T3D in vitro. Isolated C26 liver tumors were established in syngenic immunocompetent BALB/c mice by intrahepatic injection. Reovirus T3D therapy was given as a single intratumoral injection in control mice and in cyclosporin A-treated immunosuppressed mice. Tumor growth was analyzed over time by non-invasive bioluminescence imaging. The outgrowth of established CRC liver metastases in immunocompetent mice was efficiently but temporarily inhibited with a single injection of Reovirus T3D. Immunosuppression with cyclosporin A markedly increased and prolonged the therapeutic effect and allowed complete Reovirus T3D-induced tumor eradication in a subpopulation of the mice. We conclude that Reovirus T3D is an effective therapeutic agent against established C26 colorectal liver metastases and that immunosuppression enhances treatment efficacy.

Differential Notch and TGFβ Signaling in Primary Colorectal Tumors and Their Corresponding Metastases

Background: Loss of epithelial morphology and the acquisition of mesenchymal characteristics may contribute to metastasis formation during colorectal tumorigenesis. The Wnt, Notch and TGFβ signaling pathways control tissue homeostasis and tumor development in the gut. The relationship between the activity of these pathways and the expression of epithelial and mesenchymal markers was investigated in a series of primary colorectal tumors and their corresponding metastases. Methods: Tissue samples of primary colorectal tumors, normal colonic mucosa, and regional and systemic metastases were processed for immunohistochemistry in a tissue microarray format. The expression of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin) markers was related to markers of Wnt (β-catenin), Notch (HES1) and TGFβ (phospho-SMAD2) signalling. In addition, the KRAS mutation status was assessed. Results: When compared to normal mucosa, primary colorectal tumors showed a marked increase in the levels of cytoplasmic vimentin and nuclear β-catenin, phospho-SMAD2 and HES1. Increased vimentin expression correlated with the presence of oncogenic KRAS and with nuclear β-catenin. The corresponding liver, lymph node, brain and lung metastases did not express vimentin and displayed significantly lower levels of nuclear phospho-SMAD2 and HES1, while retaining nuclear β-catenin. Conclusions: Primary colorectal carcinomas display aberrant expression of vimentin, and have activated Notch and TGFβ signaling pathways. Surprisingly, many regional and distant metastases have lost nuclear HES1 and pSMAD2, suggesting that the activity of the Notch and TGFβ pathways is reduced in secondary colorectal tumors.

Sensitization to Apoptosis Underlies KrasD12-Dependent Oncolysis of Murine C26 Colorectal Carcinoma Cells by Reovirus T3D

ABSTRACT Reovirus T3D is an oncolytic agent that preferentially targets tumor cells expressing an activated Ras oncogene. Ras signaling interferes with the cellular stress response that inhibits translation of reovirus RNAs. Murine C26 colorectal carcinoma cells express a mutant KrasD12 gene. Reovirus T3D efficiently kills C26 cells, but not C26 cells in which the KrasD12 mRNA is stably repressed by expression of KrasD12-directed short-hairpin RNAs. Surprisingly, neither reovirus T3D protein synthesis nor T3D virus yields were suppressed by deletion of KrasD12. Rather, reovirus-induced tumor cell apoptosis was completely abrogated as a result of Kras knockdown. We conclude that sensitization of C26 tumor cells to reovirus-induced apoptosis underlies the Ras dependency of reovirus T3D oncolysis.

Transient infection of freshly isolated human colorectal tumor cells by reovirus T3D intermediate subviral particles

Reovirus T3D preferentially kills tumor cells expressing Ras oncogenes and has shown great promise as an anticancer agent in various preclinical tumor models. Here, we investigated whether reovirus can infect and kill tumor cell cultures and tissue fragments isolated from resected human colorectal tumors, and whether this was affected by the presence of endogenous oncogenic KRAS. Tissue fragments and single-cell populations isolated from human colorectal tumor biopsies were infected with reovirus virions or with intermediate subviral particles (ISVPs). Reovirus virions were capable of infecting neither single-cell tumor cell populations nor small fragments of intact viable tumor tissue. However, infection of tumor cells with ISVPs resulted in transient viral protein synthesis, irrespective of the presence of oncogenic KRAS, but this did not lead to the production of infectious virus particles, and tumor cell viability was largely unaffected. ISVPs failed to infect intact tissue fragments. Thermolysin treatment of tumor tissue liberated single cells from the tissue and allowed infection with ISVPs, but this did not result in the production of infectious virus particles. Immunohistochemistry on tissue microarrays showed that junction adhesion molecule 1, the major cellular reovirus receptor, was improperly localized in the cytoplasm of colorectal tumor cells and was expressed at very low levels in liver metastases. This may contribute to the observed resistance of tumor cells to reovirus T3D virions. We conclude that infection of human colorectal tumor cells by reovirus T3D requires processing of virions to ISVPs, but that oncolysis is prevented by a tumor cell response that aborts viral protein synthesis and the generation of infectious viral particles, irrespective of KRAS mutation status.

Dual effect of KrasD12 knockdown on tumorigenesis: increased immune-mediated tumor clearance and abrogation of tumor malignancy

Activating mutations in the human KRAS proto-oncogene are acquired during the earliest stages of colorectal cancer development. If mutant KRAS is to be used as a target for therapy in colorectal cancer, tumor growth should depend on its continued presence. Here, we report that stable knockdown of KrasD12 in murine C26 colorectal cancer cells by RNA interference resulted in loss of transformed properties in vitro. The incidence of subcutaneous tumor formation was reduced by 60% and the lag time was increased sevenfold. KrasD12-knockdown tumors grew noninvasively and did not cause morbidity. Remarkably, some of the KrasD12-knockdown tumors regressed spontaneously, which rendered these mice resistant to parental C26 tumor growth. In immune-deficient hosts, the incidence of tumor formation by KrasD12-knockdown cells was 100%. None of these tumors regressed spontaneously. We conclude that the reduced incidence of tumor formation by KrasD12-knockdown cells is due to tumor cell clearance by the host immune system, but not to an intrinsic inability of these cells to grow out as tumors. Interestingly, KrasD12 knockdown resulted in increased production of interleukin 18 (Il-18), an immune-stimulatory cytokine that has been implicated in limiting colorectal tumor formation. Thus, mutant KrasD12 suppresses Il-18 production in colorectal tumor cells, which may contribute to evasion of the local immune system during tumor development.

Is there an end of the “learning curve” of endoscopic totally extraperitoneal (TEP) hernia repair?

BackgroundAn important challenge of totally extraperitoneal (TEP) hernia repair is the learning curve. The European guidelines suggest that the learning curve ranges between 50 and 100 procedures, with the first 30–50 being critical. Others suggest that optimal outcomes are achieved after 200 or more TEP procedures.MethodsAll TEP repairs performed between 2005 and 2009 were included in this study. The effect of (surgeon) expertise on perioperative complications, conversion to open anterior repair, and operative time was assessed to evaluate the extent of the learning curve of TEP repair.ResultsIntraoperative complications occurred in <1xa0% of the 3,432 patients and postoperative complications were observed in 243 (7xa0%) patients. With a median follow-up of 2xa0years after TEP, 19 patients (0.55xa0%) had a recurrence. During the study period, at the end of which all four surgeons had treated 900–1,000 patients, intraoperative complications and recurrences did not decline. On the other hand, the median operative time decreased from 30 to 20 min (pxa0<xa00.001). The conversion rate (1.6–0.2xa0%, pxa0=xa00.018) and postoperative complication rate (11.6–4.2xa0%, pxa0<xa00.001) also declined. The decline was observed for all four surgeons, irrespective of their initial expertise with TEP. The largest decrease in the conversion rate was seen after at least 250 TEP procedures; the postoperative complication rate and operative time showed a linear and significant decline throughout the study period. A more or less “steady state” was observed after approximately 450 procedures per surgeon.ConclusionsEven after more than 400 individually performed TEP procedures, there is progress in reducing the conversion rate, the incidence of short-term postoperative complications, and operative time, indicating a rather long learning curve.

Female ‘groin’ hernia: totally extraperitoneal (TEP) endoscopic repair seems the most appropriate treatment modality

BackgroundAbout 30% of all female ‘groin’ hernias are femoral hernias, although often only diagnosed during surgery. A Lichtenstein repair though, as preferred treatment modality according to guidelines, would not diagnose and treat femoral hernias. Totally extraperitoneal (TEP) hernia repair, however, offers the advantage of being an appropriate modality for the diagnosis and subsequent treatment of both inguinal and femoral hernias. TEP therefore seems an appealing surgical technique for women with groin hernias.MethodsThis study included all female patients ≥18xa0years operated for a groin hernia between 2005 and 2009.ResultsA total of 183 groin hernias were repaired in 164 women. TEP was performed in 85% of women; the other 24 women underwent an open anterior (mesh) repair. Peroperatively, femoral hernias were observed in 23% of patients with primary hernias and 35% of patients with recurrent hernias. There were 30 cases (18.3%) of an incorrect preoperative diagnosis. Peroperatively, femoral hernias were observed in 17.3% of women who were diagnosed with an inguinal hernia before surgery. In addition, inguinal hernias were found in 24.0% of women who were diagnosed with a femoral hernia preoperatively. After a follow-up of 25xa0months, moderate to severe (VAS 4-10) postoperative pain was reported by 8 of 125 patients (6.4%) after TEP and 5 of 23 patients (21.7%) after open hernia repair (Pxa0=xa00.03). Five patients had a recurrent hernia, two following TEP (1.4%) and three following open anterior repair (12.5%, Pxa0=xa00.02). Two of these three patients presented with a femoral recurrence after a previous repair of an inguinal hernia.ConclusionFemoral hernias are common in women with groin hernias, but not always detected preoperatively; this argues for the use of a preperitoneal approach. TEP hernia repair combines the advantage of a peroperative diagnosis and subsequent appropriate treatment with the known good clinical outcomes.