Franz Rödel

Survivin as a Radioresistance Factor, and Prognostic and Therapeutic Target for Radiotherapy in Rectal Cancer

Apoptosis levels have been shown to predict tumor response to preoperative radiochemotherapy in rectal cancer. Recently, the prominent role of survivin, a structurally unique member of the inhibitor of apoptosis protein family, has been shown in colorectal cancer tumorigenesis and prognosis. In this study, we investigated whether survivin plays a direct role in mediating radiation resistance. We used short interfering RNA molecules to decrease survivin in radioresistant SW480 and intermediately radioresistant HCT-15 colorectal cancer cells. This resulted in a significant decrease of survivin mRNA and protein expression with a maximum at 24 to 48 hours after transfection. If irradiated during this sensitive period, an increased percentage of apoptotic cells and an increased caspase 3/7 activity in parallel with a decreased cell viability and a reduced clonogenic survival was shown. These effects were more pronounced in the radioresistant SW480 cell line with a radiation-induced cytotoxicity enhancement factor at 10% and 50% survival of 1.8 to 2.2 for SW480 and 1.5 to 1.7 for HCT-15, respectively. Furthermore, transfection with survivin short interfering RNA increased levels of G2-M arrest and levels of DNA double-strand breaks in irradiated cells. These observations indicate that cell cycle and DNA repair mechanisms may be associated with apoptosis induction in tumor cells that are otherwise resistant to killing by radiation. In a translational study of 59 patients with rectal cancer treated with a combination of radiotherapy and chemotherapy, increased survivin expression was inversely related to the levels of apoptosis, and was also associated with a significantly higher risk of a local tumor recurrence.

Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor☆

BACKGROUND Spontaneous apoptosis has been shown to predict tumor response to radiochemotherapy in rectal cancer in vivo. It remains to be elucidated, however, which genetic profile determines whether a tumor is more or less prone to apoptosis. Recently, a novel member of the inhibitor of apoptosis protein family, designated survivin, was identified. We investigated the impact of survivin expression on tumor cell apoptosis in three colorectal cell lines of different intrinsic radiosensitivities. METHODS AND MATERIALS Survivin protein expression was measured by Western blot analysis, and survivin mRNA expression by quantitative TaqMan reverse transcription polymerase chain reaction, both in untreated cell and after irradiation with 2 and 8 Gy. The expression profile was then correlated to spontaneous and radiation-induced apoptosis (Tunel-Assay, DAPI-staining) in three colorectal cell lines of low (SW 480), intermediate (HCT-15), and high radiosensitivity (SW 48), as determined by the colony-forming assay. RESULTS In vitro analysis revealed higher spontaneous and higher radiation-induced apoptosis rates in the radiosensitive line (SW 48), as compared with the more resistant line (SW 480). In Western blot analysis and in TaqMan analysis, SW 480 was characterized by a higher spontaneous expression and a pronounced induction of survivin 48 h after irradiation, whereas survivin expression was low when untreated and not increased after irradiation in the most radiosensitive line SW 48. HCT-15 was intermediate, both with respect to the level of survivin mRNA and protein expression. CONCLUSION The inverse correlation of survivin-expression with spontaneous and radiation-induced apoptosis suggests that survivin is an important inhibitor of apoptosis in colorectal cancer cell lines. Analysis of survivin mRNA or protein expression may therefore provide predictive information on radio- and chemoresistance of individual colorectal tumors.

Apoptosis as a cellular predictor for histopathologic response to neoadjuvant radiochemotherapy in patients with rectal cancer.

BACKGROUND Tumor shrinkage by preoperative radiochemotherapy (RCT) can markedly improve surgery in locally advanced (T4) rectal cancer with clear resection margins and may enable sphincter preservation in low-lying tumors. However, tumor response varies considerably, even among tumors treated according to the same protocol. If one is able to identify patients with highly radio-responsive tumors at the time of diagnosis, a selective and individualized policy of preoperative RCT might be pursued. METHODS The apoptotic index (AI), Ki-67, p53, and bcl-2 were evaluated by immunohistochemistry on pretreatment biopsies from 44 patients treated uniformly according to a prospective neoadjuvant RCT protocol (CAO/AIO/ARO-94). Treatment response was assessed histopathologically in the resected surgical specimen, using a five-point grading system. Expression of each marker was correlated with tumor response and relapse-free survival after curative surgery. RESULTS Tumors with complete (n = 3) or good (n = 28) response to RCT showed significantly higher pretreatment levels of apoptosis (mean AI: 2.06%) than tumors with moderate (n = 7), minimal (n = 5), or no regression (n = 1) from RCT (AI: 1.44%, p = 0.003). The AI was significantly related to Ki-67 (p = 0.05), but not to p53 and bcl-2 status. Tumor regression and AI best predicted relapse-free survival after combined modality treatment and curative surgery. CONCLUSION Spontaneous apoptosis in rectal cancer may serve as an important predictor of tumor regression from RCT in rectal cancer and as a significant prognosticator of relapse-free survival. Thus, this molecular marker may finally help to tailor therapy with regard to (neo-) adjuvant treatment of rectal cancer.

Nuclear export is essential for the tumor-promoting activity of survivin

Survivin appears to function as an apoptosis inhibitor and a regulator of cell division during development and tumorigenesis. Here we report the molecular characterization of the nucleocytoplasmic transport of survivin and its potential implications for tumorigenesis. We identified an evolutionary conserved Crm1‐dependent nuclear export signal (NES) in survivin. In dividing cells, the NES is essential for tethering survivin and the survivin/Aurora‐B kinase complex to the mitotic machinery, which in turn appears to be essential for proper cell division. In addition, export seems to be required for the cytoprotective activity of survivin, as export‐deficient survivin fails to protect tumor cells against chemo‐and radiotherapy‐induced apoptosis. These findings appear to be clinically relevant since preferential nuclear localization of survivin correlated with enhanced survival in colorectal cancer patients. Targeting survivins nuclear export by the application of NES‐specific antibodies promoted its nuclear accumulation and inhibited its cytoprotective function. We demonstrate that nuclear export is essential for the biological activity of survivin and promote the identification of molecular decoys to specifically interfere with survivins nuclear export as potential anticancer therapeutics. Knauer, S. K., Krämer, O. H., Knösel, T., Engels, K., Rödel, F., Kovács, A. F., Dietmaier, W., Klein‐Hitpass, L., Habtemichael, N., Schweitzer, A., Brieger, J., Rödel, C., Mann, W., Petersen, I., Heinzel, T., Stauber, R. H. Nuclear export is essential for the tumor‐promoting activity of survivin. FASEB J. 21, 207–216 (2007)

Radiobiological mechanisms in inflammatory diseases of low-dose radiation therapy

Purpose: Whereas X-irradiation with high doses is established to exert pro-inflammatory effects, low-dose radiotherapy (LD-RT) with single fractions below 1.0 Gy and a total dose below 12 Gy is clinically well known to exert anti-inflammatory and analgesic effects on several inflammatory diseases and painful degenerative disorders. Experimental studies to confirm the effectiveness, the empirical dose and fractionation schemes, and the underlying radiobiological mechanisms are still fragmentary. Method: The anti-inflammatory efficiency of LD-RT was confirmed in several experimental in vitro and in vivo models. Results: In vitro studies revealed a variety of mechanisms related to the anti-inflammatory effect, in particular the modulation of cytokine and adhesion molecule expression on activated endothelial cells and leukocytes, and of nitric oxide (NO) production and oxidative burst in activated macrophages and native granulocytes. Conclusion: Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. It is, therefore, reasonable to assume that further molecular pathways and cellular components contribute to the anti-inflammatory effect of LD-RT. This review discusses data and models revealing aspects of the mechanisms underlying the anti-inflammation induced by low doses of X-irradiation and may serve as a basis for systematic analyses, necessary to optimize LD-RT in clinical practice.

High Survivin Expression is Associated with Reduced Apoptosis in Rectal Cancer and May Predict Disease-Free Survival after Preoperative Radiochemotherapy and Surgical Resection

Background: Spontaneous apoptosis has been shown to predict tumor response to preoperative radiochemotherapy in rectal cancer. It remains to be elucidated, however, which genetic profile determines whether a tumor is more or less prone to apoptosis. Recently, a novel member of the inhibitor of apoptosis family, designated survivin, was identified. In the present study , we investigated the impact of survivin on tumor cell apoptosis and the risk to develop distant metastases or local failure after preoperative radiochemotherapy and surgical resection. Patients and Methods: The expression of survivin, p53, bcl-2 and the apoptotic index was evaluated by immunochistochemistry on pretreatment biopsies of 54 patients with locally advanced adenocarcinoma of the rectum. Survivin expression was correlated to clinical and histopathological markers, the levels of spontaneous apoptosis, p53 and bcl-2, as well as to disease-free survival, 5-year rates of local failure and distant disease after preoperative radiochemotherapy and surgical resection. Results: Survivin expression inversely correlated with the apoptotic index: High survivin expression was found in 56% of rectal carcinoma biopsies with a median apoptotic index of 1.22%. Conversely, low survivin expression in tumor cells was associated with a high median apoptotic index (2.29%, p=0.0001). High survivin expression also segregated with bcl-2 overexpression (65% bcl-2+ in tumors with high survivin expression as compared to 35% bcl-2+ in tumors with low survivin expression), but the difference was not statistically significant (p=0.1). Low survivin expression was significantly related to an increased disease-free survival rate (77% vs 18% at 5 years in tumors with high survivin expression, p=0.02) and to a redued risk for distant metastases (18% vs 78% at 5 years in tumors with high survivin expression, p=0.05) and local failure (6% vs 37% at 5 years in tumors with high survivin expression, p=0.07). Conclusion: An inverse correlation between survivin expression and the level of spontaneous apoptosis in pretreatment biopsies suggests that survivin strongly inhibits tumor cell apoptosis in rectal cancer. Survivin expression may provide a novel predictive indicator for disease-free survival after preoperative radiochemotherapy and surgical resection in rectal cancer.Hintergrund: Für die spontane Apoptoserate konnte eine prädiktive Bedeutung für die Tumorantwort nach präoperativer Radiochemotherapie beim Rektumkarzinom gezeigt werden. Bisher ist nicht geklärt, welches genetische Profil die Apoptosefähigkeit eines Tumors bestimmt. Kürzlich wurde das Protein Survivin als ein neues Mitglied der Inhibitor-of-Apoptosis-Familie identifiziert. In der vorliegenden Studie wurd der Einfluss von Survivin auf die Apoptoserate sowie das Fernmetastasenrisiko und die Rate lokoregionärer Rezidive nach neoadjuvanter Radiochemotherapie und Operation des Rektumkarzinoms untersucht. Patienten und Methoden: Die Expression von Survivin, p53, bcl-2 und die Apoptsoerate wurden immunhistochemisch in prätherapeutischen Biopsien von 54 Patienten mit lokal fortgeschrittenen Adenokarzinomen des Rektums bestimmt, die einheitich nach einem neoadjuvanten Radiochemotherapieprotokoll behandelt wurden. Die Survivin-Expression wurde mit klinischen und histopathologischen Markern, dem p53- und bcl-2-Index, dem Apoptoseindex, dem krankheitsfreien Überleben sowie dem Auftreten von Fernmetastasen oder lokoregionären Rezidiven korreliert. Ergebnisse: Die Survivin-Expression korrelierte invers mit dem Apoptoseindex: Bei hoher Survivin-Expression (56% aller Tumoren) betrug der mediane Apoptoseindex 1,22%, bei niedriger Survivin-Expression 2,29% (p=0,0001). Eine erhöhte Survivin-Expression war auch mit einer bcl-2-Überexpression assoziiert (65% bcl-2+ in Tumoren mit hoher Survivin-Expression, 35% bcl-2+ in Tumoren mit niedriger Survivin-Expression, p=0,1). Bei niedriger Survivin-Expression betrug das krankheitsfreie Überleben nach 5 Jahren 77%, bei hoher Survivin-Expression 18% (p=0,02). Die Fernmetastasenrate und die Rate an lokoregionären Rezidiven betrugen nach 5 Jahren bei Tumoren mit niedriger bzw. hoher Survivin-Expression: 18% vs. 78 % (p=0,05) bzw. 6% vs. 37% (p=0,07). Schlussfolgerung: Die signifikant inverse Korrelation zwischen der Survivin-Expression und der spontanen Apoptoserate in prätherapeutischen Biopsien weist darauf hin, dass Survivin beim Rektumkarzinom die Apoptose der Tumorzellen inhibiert. Die Survivin-Expression kann als neuer prädiktiver Indikator für das krankheitsfreie Überleben nach präoperativer Radiochemotherapie und Operation gelten.

Tumour-infiltrating lymphocytes predict response to definitive chemoradiotherapy in head and neck cancer

Background:We aimed to investigate the prognostic value of tumour-infiltrating lymphocytes’ (TILs) expression in pretreatment specimens from patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).Methods:The prevalence of CD3+, CD8+, CD4+ and FOXP3+ TILs was assessed using immunohistochemistry in tumour tissue obtained from 101 patients before CRT and was correlated with clinicopathological characteristics as well as local failure-free- (LFFS), distant metastases free- (DMFS), progression-free (PFS) and overall survival (OS). Survival curves were measured using the Kaplan–Meier method, and differences in survival between the groups were estimated using the log-rank test. Prognostic effects of TIL subset density were determined using the Cox regression analysis.Results:With a mean follow-up of 25 months (range, 2.3–63 months), OS at 2 years was 57.4% for the entire cohort. Patients with high immunohistochemical CD3 and CD8 expression had significantly increased OS (P=0.024 and P=0.028), PFS (P=0.044 and P=0.047) and DMFS (P=0.021 and P=0.026) but not LFFS (P=0.90 and P=0.104) in multivariate analysis that included predictive clinicopathologic factors, such as age, sex, T-stage, N-stage, tumour grading and localisation. Neither CD4 nor FOXP3 expression showed significance for the clinical outcome. The lower N-stage was associated with improved OS in the multivariate analysis (P=0.049).Conclusion:The positive correlation between a high number of infiltrating CD3+ and CD8+ cells and clinical outcome indicates that TILs may have a beneficial role in HNSCC patients and may serve as a biomarker to identify patients likely to benefit from definitive CRT.

SURVIVIN ANTISENSE OLIGONUCLEOTIDES EFFECTIVELY RADIOSENSITIZE COLORECTAL CANCER CELLS IN BOTH TISSUE CULTURE AND MURINE XENOGRAFT MODELS

PURPOSE Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. METHODS AND MATERIALS SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, gammaH2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. RESULTS In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G(2)/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. CONCLUSION Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

Apoptosis, p53, bcl-2, and Ki-67 in invasive bladder carcinoma: possible predictors for response to radiochemotherapy and successful bladder preservation☆

PURPOSE Several groups have reported the value of bladder preservation by a combined treatment protocol, including transurethral resection (TUR-B) and radiochemotherapy (RCT). As more experience is acquired with organ-sparing treatment, patient selection should be optimized. The purpose of this study was to investigate the role of several biologic markers that may predict response to RCT in muscle-invasive bladder carcinoma. METHODS AND MATERIALS The apoptotic index (AI), Ki-67, p53, and bcl-2 were evaluated by immunohistochemistry on pretreatment biopsies from 70 patients treated for invasive bladder cancer by TUR-B and RCT. Expression of each marker was correlated with initial response, local control, and cancer-specific survival with preserved bladder. An exploratory multivariate analysis was also performed that included clinical and immunohistochemical variables. RESULTS A high AI (> median = 1.6%) and a high Ki-67 index (> median = 8.8%), but not the p53- and bcl-2 expression, were significantly related to initial complete response (CR) and local control with preserved bladder after 5 years. When the AI and Ki-67 expression were considered simultaneously, the association with initial CR (p < 0. 001), local control (p = 0.0002), and cancer-specific survival with preserved bladder (p = 0.008) was highly significant. In an exploratory multivariate analysis (final model), only AI, Ki-67, and the combined AI/Ki-67 variable retained significance for local control with preserved bladder at 5 years. CONCLUSION Patients with a high spontaneous AI and a high pretreatment Ki-67 index should be considered preferentially for treatment with RCT, whereas tumors with low proliferation and low levels of apoptosis are less likely to respond to RCT.

Stromal SPARC expression and patient survival after chemoradiation for non-resectable pancreatic adenocarcinoma

Purpose Pancreatic stellate cells (PSC) drive desmoplasia in pancreatic cancer. Our study analyzed both tumor and PSC, since interaction of these cell types may promote tumor progression. Experimental design Immunohistochemical expression of SPARC, CTGF, TGF-β1, phospho-Akt, survivin and α-SMA was analyzed prior to chemoradiation in 58 locally advanced pancreatic cancer (LAPC) biopsy specimens. Fisher’s exact test served to detect associations between tumor and PSC expression of markers. Kaplan-Meier analysis and multivariate analysis were used to evaluate the association of marker expression with overall survival. SPARC expression was analyzed in human pancreatic cancer cells (Panc-1) and in human PSC (hPSC), and the effect of SPARC on the invasion of Panc-1 cells was measured in monoculture or in coculture with hPSC. Results SPARC was expressed predominantly in the peritumoral and distal stroma. SPARC in distal stroma correlated inversely with overall survival of the patients with LAPC (p=0.013) with a relative hazard of 2.23 (95% CI, 1.05 to 4.72; P=0.036). TGF-β1 in the tumor was also a negative prognostic factor (P=0.03). Within the tumor cells, phospho-Akt correlated with TGF-β1, SPARC and survivin. Tumor phospho-Akt correlated with stroma phospho-Akt, tumor TGF-β1 correlated with stroma TGF-β1 and α-SMA, tumor survivin correlated with stroma survivin and distal SPARC. Within the stroma, SPARC and TGF-β1 correlated with α-SMA. Peritumoral SPARC correlated with distal SPARC. In vitro, SPARC was highly expressed in hPSC but not in Panc-1 cells. Exogenous SPARC did not change radiation resistance but increased the invasion of Panc-1 cells both in monoculture and in coculture with hPSC. Conclusions Our hypothesis of a detrimental effect of PSC on patient survival in LAPC after chemoradiation is supported by the inverse correlation of SPARC in distal stromal cells with patients survival. Furthermore in vitro data indicate that paracrine SPARC from PSC increases the invasion of pancreatic cancer cells.