Tobias Bartscht

The Src family kinase inhibitors PP2 and PP1 effectively block TGF-beta1-induced cell migration and invasion in both established and primary carcinoma cells

PurposeWe have previously demonstrated that in pancreatic ductal adenocarcinoma (PDAC)-derived cell lines, the common Src family kinase inhibitors PP2 and PP1 effectively inhibited morphologic alterations associated with TGFβ1-mediated epithelial-to-mesenchymal transition (EMT) by blocking the kinase activity of the TGF-β type I receptor ALK5 rather than Src (Ungefroren et al. in Curr Cancer Drug Targets 11:524, 2011). In this report, the ability of PP2 and PP1, the more specific Src inhibitor SU6656, and the ALK5 inhibitor SB431542 to functionally block TGF-β1-dependent EMT and cell motility in established PDAC (Panc-1, Colo 357) and primary NSCLC (Tu459) cell lines were investigated.MethodsThe effects of PP2, PP1, SU6656, and SB431542 on TGF-β1-dependent cell scattering/EMT, cell migration/invasion, and expression of invasion-associated genes were measured by using the real-time cell analysis assay on the xCELLigence system and quantitative real-time RT-PCR, respectively.ResultsIn all three cell lines tested, PP1, PP2, and SB431542 effectively blocked TGF-β1-induced cell scattering/EMT, migration, and invasion and in Colo 357 cells inhibited the induction of the invasion-associated MMP2 and MMP9 genes. In contrast, SU6656 only blocked TGF-β1-induced invasion in Panc-1 and Tu459 but not Colo 357 cells. PP1, and to a greater extent PP2, also inhibited the high spontaneous migratory activity of Panc-1 cells expressing a kinase-active ALK5 mutant.ConclusionsThese data provide evidence that PP2 and PP1 are powerful inhibitors of TGF-β-induced cell migration and invasion in vitro and directly target ALK5. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics to prevent metastatic spread in late-stage PDAC and NSCLC.

Prognostic role of the number of involved extraspinal organs in patients with metastatic spinal cord compression

OBJECTIVE This study was investigated the prognostic role of the number of involved extraspinal organs in the survival of patients with metastatic spinal cord compression (MSCC). METHODS Data of 552 patients treated with 30Gy in 10 fractions of radiotherapy (RT) alone for MSCC were retrospectively analyzed. In addition to the number of involved extraspinal organs, eight potential prognostic factors were investigated including age, gender, Eastern Cooperative Oncology Group performance score (ECOG-PS), primary tumor type, number of involved vertebrae, interval from cancer diagnosis to RT, pre-RT ambulatory status, and time developing motor deficits. RESULTS The 6-month survival rates for the involvement of 0, 1, 2, 3, and ≥4 extraspinal organs were 88%, 55%, 30%, 13%, and 12%, respectively (P<0.001). In the multivariate analysis, number of involved extraspinal organs maintained significance (risk ratio 1.61; 95%-confidence interval 1.47-1.77; P<0.001). On multivariate analysis, gender (P=0.017), ECOG-PS (P<0.001), primary tumor type (P<0.001), interval from cancer diagnosis to RT (P<0.001), pre-RT ambulatory status (P<0.001), and time developing motor deficits (P<0.001) were also independent predictors for survival. CONCLUSIONS The number of involved extraspinal organs is a new and independent prognostic factor in patients with MSCC and should be considered in future clinical trials.

Dasatinib blocks transcriptional and promigratory responses to transforming growth factor-beta in pancreatic adenocarcinoma cells through inhibition of Smad signalling: implications for in vivo mode of action

BackgroundWe have previously shown in pancreatic ductal adenocarcinoma (PDAC) cells that the SRC inhibitors PP2 and PP1 effectively inhibited TGF-β1-mediated cellular responses by blocking the kinase function of the TGF-β type I receptor ALK5 rather than SRC. Here, we investigated the ability of the clinically utilised SRC/ABL inhibitor dasatinib to mimic the PP2/PP1 effect.MethodsThe effect of dasatinib on TGF-β1-dependent Smad2/3 phosphorylation, general transcriptional activity, gene expression, cell motility, and the generation of tumour stem cells was measured in Panc-1 and Colo-357 cells using immunoblotting, reporter gene assays, RT-PCR, impedance-based real-time measurement of cell migration, and colony formation assays, respectively.ResultsIn both PDAC cell lines, dasatinib effectively blocked TGF-β1-induced Smad phosphorylation, activity of 3TPlux and pCAGA(12)-luc reporter genes, cell migration, and expression of individual TGF-β1 target genes associated with epithelial-mesenchymal transition and invasion. Moreover, dasatinib strongly interfered with the TGF-β1-induced generation of tumour stem cells as demonstrated by gene expression analysis and single cell colony formation. Dasatinib also inhibited the high constitutive migratory activity conferred on Panc-1 cells by ectopic expression of kinase-active ALK5.ConclusionsOur data suggest that the clinical efficiency of dasatinib may in part be due to cross-inhibition of tumour-promoting TGF-β signalling. Dasatinib may be useful as a dual TGF-β/SRC inhibitor in experimental and clinical therapeutics to prevent metastatic spread in late-stage PDAC and other tumours.

ERG expression in multiple myeloma—A potential diagnostic pitfall

INTRODUCTION ERG expression has been described as a frequent event in prostate cancer indicating poor prognosis and promoting oncogenesis. It has also been demonstrated in Ewings sarcoma, acute myeloid leukemia and acute T-lymphoblastic leukemia but could not be found in other epithelial tumors, Hodgkins or Non-Hodgkins lymphoma. We aimed to analyze ERG expression in multiple myeloma, following an index case of a patient with metastases of unknown origin in the spine strongly expressing ERG, which were thought to be of prostatic origin but turned out to be plasmacytic lesions. MATERIAL AND METHODS We subsequently selected 12 formalin-fixed, paraffin-embedded tissue samples of multiple myeloma from our archives and performed immunohistochemical staining for ERG. RESULTS All 12 analyzed cases showed strong nuclear expression of ERG in >90% of tumor cells (myeloma cells). CONCLUSIONS This report highlights a potential and critical diagnostic pitfall in biopsy specimens where morphology is only of limited assistance in reaching the correct diagnosis. It urges pathologists to exercise caution in cases where strong ERG-positivity implicates the presence of a prostatic neoplasia and illustrates the need for further immunohistochemical examination.

A new instrument for estimating the survival of patients with metastatic epidural spinal cord compression from esophageal cancer

Abstract Background. This study was initiated to create a predictive instrument for estimating the survival of patients with metastatic epidural spinal cord compression (MESCC) from esophageal cancer. Methods. In 27 patients irradiated for MESCC from esophageal cancer, the following nine characteristics were evaluated for potential impact on survival: age, gender, Eastern Cooperative Oncology Group (ECOG) performance score, histology, number of involved vertebrae, ambulatory status before irradiation, further bone metastases, visceral metastases, and dynamic of developing motor deficits before irradiation. In addition, the impact of the radiation regimen was investigated. According to Bonferroni correction, p-values of < 0.006 were significant representing an alpha level of < 0.05. Results. ECOG performance score (p < 0.001), number of involved vertebrae (p = 0.005), and visceral metastases (p = 0.004) had a significant impact on survival and were included in the predictive instrument. Scoring points for each characteristic were calculated by dividing the 6-months survival rates (in %) by 10. The prognostic score for each patient was obtained by adding the scoring points of the three characteristics. The prognostic scores were 4, 9, 10, 14 or 20 points. Three prognostic groups were formed, 4 points (n = 11), 9-14 points (n = 12) and 20 points (n = 4). The corresponding 6-months survival rates were 0%, 33% and 100%, respectively (p < 0.001). Median survival times were 1 month, 5 months and 16.5 months, respectively. Conclusions. This new instrument allows the physician estimate the 6-months survival probability of an individual patient presenting with MESCC from esophageal cancer. This is important to know for optimally personalizing the treatment of these patients.

Predictors of survival in patients with brain metastases from gastric cancer

This study aims to identify predictors of survival and contribute to treatment personalization in patients with brain metastases from gastric cancer. Twelve patients received whole-brain radiotherapy (WBRT), four stereotactic radiosurgery and six neurosurgery plus WBRT. Treatment regimen, age, gender, Eastern Cooperative Oncology Group (ECOG) performance score, tumor site, number of brain metastases, extra-cranial metastases and interval between cancer diagnosis and brain metastases were evaluated for survival. On univariate analyses, more intensive treatment (p=0.003), ECOG-score 0-1 (p<0.001), cardiac location (p=0.025) and single brain metastasis (p=0.023) were associated with better survival. On multivariate analysis, ECOG-score maintained significance (p<0.001). Patients with all three positive factors on univariate analysis had a 12-month survival rate of 100%, patients with three negative factors a 3-month survival rate of 0%. Predictors of survival were identified that can guide physicians selecting personalized treatment approaches for patients with brain metastases from gastric cancer.

Inhibition of TGF-β Signaling in Tumor Cells by Small Molecule Src Family Kinase Inhibitors

In a series of studies carried out over the last couple of years in various cell types, it was observed that the experimentally used Src family kinase inhibitors PP1 and PP2 and the clinically used Src/Abl inhibitors AZM475271 and dasatinib are potent inhibitors of TGF-β mediated cellular responses such as Smad and p38 mitogen-activated protein kinase phosphorylation, Smad-dependent transcriptional activation, growth inhibition, epithelial-mesenchymal transition (EMT), and cell motility. While for PP1/PP2 it was demonstrated that these agents directly inhibit the kinase activity of the TGF-β type I receptor activin receptor-like kinase 5, the mechanism of the anti-TGF-β effect of AZM475271 and dasatinib is less clear. In contrast, the anti-TGF-β effect of yet another Src/Abl inhibitor, bosutinib, is more variable with respect to the type of the TGF-β response and the cell type affected, and lacks a clear dose-dependency. In the light of their strong anti-activin receptor-like kinase 5 kinase effect, PP1 and PP2 should not be used when studying the role of c-Src as downstream mediators in TGF-β/activin receptor-like kinase 5 signaling. On the other hand, based upon in vitro findings, it is conceivable that part of the therapeutic effects of AZM475271 and dasatinib seen in preclinical and clinical studies with solid tumors was caused by inhibition of prometastatic TGF-β rather than Src signaling. If AZM475271 and dasatinib can indeed act as dual Src / TGF-β inhibitors in vivo, this may be beneficial for prevention of metastatic disease in more advanced tumor stages.

TGF-β Signal Transduction in Pancreatic Carcinoma Cells is Sensitive to Inhibition by the Src Tyrosine Kinase Inhibitor AZM475271

BACKGROUND Earlier results from our group have shown that in pancreatic ductal adenocarcinoma (PDAC)-derived cells transforming growth factor (TGF)-β1-dependent epithelial-mesenchymal transition (EMT) and cell motility was inhibited by the Src inhibitors PP2 and PP1 both of which targeted the TGF-β receptors for inhibition. OBJECTIVE In this study we evaluated the impact of another Src inhibitor, AZM475271, on various TGF-β responses in PDAC cells. METHOD The effect of AZM475271 on TGF-β1-induced random cell migration (chemokinesis), the expression of EMT and migration/invasion-associated genes, TGF-β-induced luciferase activity, and C-terminal phosphorylation of Smad2 and Smad3 was measured in the PDAC-derived Panc-1 and Colo357 cell lines using real-time cell migration assays, quantitative real-time PCR, luciferase reporter gene assays and phosphoimmunoblotting, respectively. RESULTS AZM475271 effectively blocked TGF-β1-induced chemokinesis of Panc-1 cells in a dose-dependent fashion and inhibited the high chemokinetic activity of Panc-1 cells with ectopic expression of a constitutively active ALK5T204D mutant. AZM475271 but not another Src inhibitor, SU6656, partially relieved the suppressive effect of TGF-β1 on E-cadherin and inhibited TGF-β1-induced upregulation of the MMP2, MMP9, N-cadherin and vimentin genes, activity of a TGF-β1-dependent reporter gene, and activation of Smad2 and Smad3. CONCLUSION Our data suggest that AZM475271 cross-inhibits tumor-promoting TGF-β signaling and may thus function as an inhibitor of both TGF-β and Src in both experimental and clinical therapies against metastatic dissemination in late-stage PDAC.

Hyperfractionated or accelerated hyperfractionated re-irradiation with ≥42 Gy in combination with paclitaxel for secondary/recurrent head-and-neck cancer

Background/Aim: Patients with secondary/ recurrent squamous cell head and neck cancer (SCCHN) have poor prognoses. Outcomes of re-irradiation with ≥42 Gy plus paclitaxel for secondary/recurrent SCCHN are herein presented. Patients and Methods: Two patients re-irradiated for secondary/recurrent SCCHN were evaluated. Patients received 44.4 Gy (2×1.2 Gy/day) or 42.0 Gy (2×1.5 Gy/day), respectively, plus concurrent paclitaxel (35 mg/m2 weekly or 20 mg/m2 twice per week). Results: One patient developed a locoregional recurrence and additional metastases at 12 months after re-irradiation and died at 13 months. The other patient developed multiple bone metastases at 103 months and died at 104 months. Acute toxicities included grade 2 anemia and mucositis in both patients. Radiation dermatitis was grade 2 in one patient and grade 3 in the other. Conclusion: Re-irradiation with 42.0-44.4 Gy given twice daily plus paclitaxel was well tolerated and achieved a favorable response. The results need to be confirmed in a prospective trial.

[A 40-year-old patient with diffuse pain in the lower backbone].

CASE REPORT A 40-year-old female patient with pancreatic cancer stage IV and multiple liver metastases is reported. Palliative chemotherapy was performed with gemcitabine monotherapy. A good control of tumor growth as well as tumor-related symptoms could be achieved for years. Finally, a rapid progression within 2 months resulted in the patients death. CONCLUSION This case report demonstrates the problems of modern pancreatic cancer therapy. The goals of therapy are cancer control and control of tumor-related symptoms. New agents and the inclusion of tumor biology and mechanism of resistance should result in individual treatment strategies.Zusammenfassung.Fallbeschreibung:Es wird über eine 40-jährige Patientin mit einem Karzinom des Pankreasschwanzes berichtet, das aufgrund multipler hepatischer Filiae bereits bei Diagnosestellung im Stadium IV vorlag. Die palliative Chemotherapie bestand aus einer Gemcitabinmonotherapie mit einer guten Tumorkontrolle sowie einer deutlichen Stabilisierung der tumorspezifischen Symptome über mehrere Jahre. Final kam es innerhalb von 2 Monaten zu einer raschen Progredienz.Schlussfolgerung:Die vorliegende Kasuistik zeigt exemplarisch den heutigen Stand der Therapie des Pankreaskarzinoms. Ziele der Therapie sind Tumorkontrolle und Beherrschung der tumorspezifischen Symptome. In zukünftige Therapiemodalitäten sollten Erkenntnisse der Tumorbiologie und Resistenzentwicklung einfließen. Auf diesem Boden sollte eine individualisierte Therapie- und Risikostratifizierung erfolgen.Abstract.Case Report:A 40-year-old female patient with pancreatic cancer stage IV and multiple liver metastases is reported. Palliative chemotherapy was performed with gemcitabine monotherapy. A good control of tumor growth as well as tumor-related symptoms could be achieved for years. Finally, a rapid progression within 2 months resulted in the patient’s death.Conclusion:This case report demonstrates the problems of modern pancreatic cancer therapy. The goals of therapy are cancer control and control of tumor-related symptoms. New agents and the inclusion of tumor biology and mechanism of resistance should result in individual treatment strategies.