Margret Rave-Fränk

A gene expression signature for chemoradiosensitivity of colorectal cancer cells.

PURPOSE The standard treatment of patients with locally advanced rectal cancers comprises preoperative 5-fluorouracil-based chemoradiotherapy followed by standardized surgery. However, tumor response to multimodal treatment has varied greatly, ranging from complete resistance to complete pathologic regression. The prediction of the response is, therefore, an important clinical need. METHODS AND MATERIALS To establish in vitro models for studying the molecular basis of this heterogeneous tumor response, we exposed 12 colorectal cancer cell lines to 3 μM of 5-fluorouracil and 2 Gy of radiation. The differences in treatment sensitivity were then correlated with the pretherapeutic gene expression profiles of these cell lines. RESULTS We observed a heterogeneous response, with surviving fractions ranging from 0.28 to 0.81, closely recapitulating clinical reality. Using a linear model analysis, we identified 4,796 features whose expression levels correlated significantly with the sensitivity to chemoradiotherapy (Q <.05), including many genes involved in the mitogen-activated protein kinase signaling pathway or cell cycle genes. These data have suggested a potential relevance of the insulin and Wnt signaling pathways for treatment response, and we identified STAT3, RASSF1, DOK3, and ERBB2 as potential therapeutic targets. The microarray measurements were independently validated for a subset of these genes using real-time polymerase chain reactions. CONCLUSION We are the first to report a gene expression signature for the in vitro chemoradiosensitivity of colorectal cancer cells. We anticipate that this analysis will unveil molecular biomarkers predictive of the response of rectal cancers to chemoradiotherapy and enable the identification of genes that could serve as targets to sensitize a priori resistant primary tumors.

Silencing of the Wnt transcription factor TCF4 sensitizes colorectal cancer cells to (chemo-) radiotherapy.

A considerable percentage of rectal cancers are resistant to standard preoperative chemoradiotherapy. Because patients with a priori-resistant tumors do not benefit from multimodal treatment, understanding and overcoming this resistance remains of utmost clinical importance. We recently reported overexpression of the Wnt transcription factor TCF4, also known as TCF7L2, in rectal cancers that were resistant to 5-fluorouracil-based chemoradiotherapy. Because Wnt signaling has not been associated with treatment response, we aimed to investigate whether TCF4 mediates chemoradioresistance. RNA interference-mediated silencing of TCF4 was employed in three colorectal cancer (CRC) cell lines, and sensitivity to (chemo-) radiotherapy was assessed using a standard colony formation assay. Silencing of TCF4 caused a significant sensitization of CRC cells to clinically relevant doses of X-rays. This effect was restricted to tumor cells with high T cell factor (TCF) reporter activity, presumably in a β-catenin-independent manner. Radiosensitization was the consequence of (i) a transcriptional deregulation of Wnt/TCF4 target genes, (ii) a silencing-induced G(2)/M phase arrest, (iii) an impaired ability to adequately halt cell cycle progression after radiation and (iv) a compromised DNA double strand break repair as assessed by γH2AX staining. Taken together, our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance. Moreover, they suggest that TCF4 is a promising molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy.

Single-Dose Gamma-Irradiation Induces Up-Regulation of Chemokine Gene Expression and Recruitment of Granulocytes into the Portal Area but Not into Other Regions of Rat Hepatic Tissue

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Role of DNA-PK in the process of aberration formation as studied in irradiated human glioblastoma cell lines M059K and M059J.

Purpose : The participation of various DNA double-strand break repair mechanisms in the formation of chromosome aberrations is not yet fully understood. To investigate particularly the role of non-homologous end-joining, we analysed the formation of radiation-induced aberrations in a DNA-protein kinase (PK CS) -proficient cell line M059K and in a deficient cell line M059J. Materials and methods : Plateau-phase M059K and M059J cells were irradiated with low doses of X-rays. Chromosome aberrations were determined as genomic yields of dicentric chromosomes and excess acentric fragments, scored in Giemsa-stained metaphases, and as partial yields of reciprocal translocations and total visible complex exchanges (complex aberrations) for chromosomes 4, 7 and 11 using the FISH method. M059K cells were also analysed in the presence of 50 µ;m wortmannin, a DNA-PK inhibitor. Results : DNA-PK-deficient cells showed a higher yield of simple stable and unstable and of complex aberrations in comparison with DNA-PK-proficient cells. The largest differences were observed for excess acentric fragments and for complex aberrations. DNA-PK inhibition by wortmannin in M059-K cells resulted in increased aberration yield in the same qualitative and quantitative manner as in M059-J cells. Conclusion : The results obtained with DNA-PK-deficient M059J cells and with DNA-PK-proficient M059K cells treated with wortmannin, an inhibitor of DNA-PK and ATM, suggest that the elimination of DNA-PK-dependent non-homologous end-joining can recruit a slow, error-prone repair process, which is DNA-PK independent and favours the increased formation of chromosome aberrations. The nature of this pathway and the way of its participation in aberration formation need further elucidation.

PALB2 mutations in German and Russian patients with bilateral breast cancer.

Since germline mutations in the PALB2 (Partner and Localizer of BRCA2) gene have been identified as breast cancer (BC) susceptibility alleles, the geographical spread and risks associated with PALB2 mutations are subject of intense investigation. Patients with bilateral breast cancer constitute a valuable group for genetic studies. We have thus scanned the whole coding region of PALB2 in a total of 203 German or Russian bilateral breast cancer patients using an approach based on high-resolution melting analysis and direct sequencing of genomic DNA samples. Truncating PALB2 mutations were identified in 4/203 (2%) breast cancer patients with bilateral disease. The two nonsense mutations, p.E545X and p.Q921X, have not been previously described whereas the two other mutations, p.R414X and c.509_510delGA, are recurrent. Our results indicate that PALB2 germline mutations account for a small, but not negligible, proportion of bilateral breast carcinomas in German and Russian populations.

STAT3 inhibition sensitizes colorectal cancer to chemoradiotherapy in vitro and in vivo.

Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers (CRCs). We have recently reported that STAT3 gene expression correlates with resistance of CRC cell lines to 5‐fluorouracil (5‐FU)‐based chemoradiotherapy (CT/RT). This is of considerable clinical importance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. To test whether STAT3 contributes to CT/RT‐resistance, we first confirmed that STAT3 protein expression correlated positively with increasing resistance. While STAT3 was not constitutively active, stimulation with interleukin‐6 (IL‐6) resulted in remarkably higher expression levels of phosphorylated STAT3 in CT/RT‐resistant cell lines. A similar result was observed when we determined IL‐6‐induced expression levels of phosphorylated STAT3 following irradiation. Next, STAT3 was inhibited in SW480 and SW837 using siRNA, shRNA and the small‐molecule inhibitor STATTIC. Successful silencing and inhibition of phosphorylation was confirmed using Western blot analysis and a luciferase reporter assay. RNAi‐mediated silencing as well as STATTIC treatment resulted in significantly decreased clonogenic survival following exposure to 3 µM of 5‐FU and irradiation in a dose‐dependent manner, with dose‐modifying factors of 1.3–2.5 at a surviving fraction of 0.37. Finally, STAT3 inhibition led to a profound CT/RT‐sensitization in a subcutaneous xenograft model, with a significantly delayed tumor regrowth in STATTIC‐treated mice compared with control animals. These results highlight a potential role of STAT3 in mediating treatment resistance and provide first proof of concept that STAT3 represents a promising novel molecular target for sensitizing resistant rectal cancers to CT/RT.

Human papillomavirus DNA load and p16INK4a expression predict for local control in patients with anal squamous cell carcinoma treated with chemoradiotherapy.

As the detection rate of HPV‐DNA in anal carcinoma commonly exceeds 90%, a comparison between sole HPV‐positive and HPV‐negative cancers with respect to treatment response following chemoradiotherapy (CRT) and long‐term oncological outcome is challenging. Against this background, we aimed to assess HPV types and HPV DNA load in formalin‐fixed paraffin‐embedded tissue (FFPE) of 95 patients treated with standard CRT for anal cancer to correlate viral load (≤/> median) with local failure, distant metastases, cancer‐specific (CSS) and overall survival (OS) rates. Various clinicopathologic parameters and the immunohistochemical marker p16INK4a were evaluated for any correlation with HPV16 DNA load and were included in uni‐ and multivariate analyses. The overall prevalence of HPV DNA was 95.8% with HPV16 monoinfection being the most commonly encountered HPV type (78.9%), followed by HPV16 and 31, 35, 39, 44, 58, 66 and 81 dual infection in 9 patients (9.5%). HPV16 DNA load was significantly associated with p16INK4a expression (p = 0.001). Patients with HPV16 DNA load ≤ median and low p16INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p = 0.023, multivariate p = 0.042; p16INK4a: univariate p = 0.021), and OS (HPV16 DNA load: univariate p = 0.02, multivariate p = 0.03). Moreover, a combined HPV16 DNA load and p16INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p = 0.019, p = 0.04 and p = 0.031). In conclusion, these data indicate that HPV16 DNA load and p16INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.

Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis

Abstract Christiansen, H., Batusic, D., Saile, B., Hermann, R. M., Dudas, J., Rave-Frank, M., Hess, C. F., Schmidberger, H. and Ramadori, G. Identification of Genes Responsive to Gamma Radiation in Rat Hepatocytes and Rat Liver by cDNA Array Gene Expression Analysis. Radiat. Res. 165, 318–325 (2006). The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.

Effects of docetaxel in combination with radiation on human head and neck cancer cells (ZMK-1) and cervical squamous cell carcinoma cells (CaSki ).

The purpose of this study was to determine, as we did for paclitaxel, the cytotoxic and radiosensitizing potential of docetaxel in human head and neck cancer cells (ZMK‐1), and in cervical squamous cell carcinoma cells (CaSki). ZMK‐1 cells were incubated with docetaxel for 3, 9 or 24 hr before irradiation and 24 hr after irradiation. CaSki cells were incubated with docetaxel 24 hr before and after irradiation. For ZMK‐1 cells, the docetaxel concentrations (0.7, 0.7 and 0.35 nM) were determined to obtain approximately equivalent cell survival at the different incubation times (3, 9 and 24 hr, respectively). For CaSki cells, the necessary concentration of docetaxel was 0.07 nM. Radiation doses were given from 0 to 7 Gy. Cell survival was measured by a standard clonogenic assay after a 9‐day incubation. Flow cytometry was used to measure the capacity of docetaxel to accumulate cells in the G2/M phase of the cell cycle. We observed a weak accumulation of cells in the G2/M phase for the ZMK‐1 cells and a pronounced accumulation for CaSki cells. For docetaxel incubation before irradiation, the isoeffect enhancement ratios for ZMK‐1 cells determined at the 37% survival level were 1.18, 2.01, and 2.40 for pre‐incubation at 3, 9 and 24 hr, respectively; for CaSki cells the ratio was 1.44. For a docetaxel incubation of 24 hr after irradiation, the isoeffect enhancement ratios determined at the 37% survival level were 1.54 and 1.17 for the ZMK‐1, and CaSki cells, respectively. A radiosensitizing effect of docetaxel could be demonstrated unambiguously in the two cell lines used. In contrast to our previously published results with paclitaxel, docetaxel seems to be a better radiosensitizer than paclitaxel.

High-Grade Acute Organ Toxicity During Preoperative Radiochemotherapy as Positive Predictor for Complete Histopathologic Tumor Regression in Multimodal Treatment of Locally Advanced Rectal Cancer*

Purpose:To test for a possible correlation between high-grade acute organ toxicity during preoperative radiochemotherapy and complete tumor regression after total mesorectal excision in multimodal treatment of locally advanced rectal cancer.Patients and Methods:From 2001 to 2008, 120 patients were treated. Preoperative treatment consisted of normofractionated radiotherapy at a total dose of 50.4 Gy, and either two cycles of 5-fluorouracil (5-FU) or two cycles of 5-FU and oxaliplatin. Toxicity during treatment was monitored weekly, and any toxicity CTC (Common Toxicity Criteria) ≥ grade 2 of enteritis, proctitis or cystitis was assessed as high-grade organ toxicity for later analysis. Complete histopathologic tumor regression (TRG4) was defined as the absence of any viable tumor cells.Results:A significant coherency between high-grade acute organ toxicity and complete histopathologic tumor regression was found, which was independent of other factors like the preoperative chemotherapy schedule. The probability of patients with acute organ toxicity ≥ grade 2 to achieve TRG4 after neoadjuvant treatment was more than three times higher than for patients without toxicity (odds ratio: 3.29, 95% confidence interval: [1.01, 10.96]).Conclusion:Acute organ toxicity during preoperative radiochemotherapy in rectal cancer could be an early predictor of treatment response in terms of complete tumor regression. Its possible impact on local control and survival is under further prospective evaluation by the authors’ working group.Ziel:Überprüfung einer möglichen Korrelation zwischen höhergradiger akuter Organtoxizität während präoperativer Radiochemotherapie und kompletter Tumorregression nach totaler mesorektaler Exzision in der multimodalen Behandlung von lokal fortgeschrittenen Rektumkarzinomen.Patienten und Methodik:Im Zeitraum von 2001 bis 2008 wurden 120 Patienten behandelt. Die präoperative Behandlung bestand aus einer normofraktionierten Radiotherapie mit einer Gesamtdosis von 50,4 Gy und entweder zwei Zyklen 5-Fluorouracil (5-FU) oder zwei Zyklen 5-FU und Oxaliplatin. Die Toxizität während der Behandlung wurde wöchentlich untersucht. Jede Toxizität ≥ Grad 2 nach CTC (Common Toxicity Criteria) in Form von Enteritis, Proktitis oder Zystitis wurde dabei als höhergradige Organtoxizität gewertet und für spätere Analysen verwendet. Bei vollständigem histopathologischen Tumoransprechen (TRG4) konnten im Operationspräparat nach neoadjuvanter Therapie keine vitalen Tumorzellen mehr identifiziert werden.Ergebnisse:Es fand sich eine signifikante Korrelation zwischen höhergradiger akuter Organtoxizität und kompletter Tumorregression, und zwar in multivariater Analyse unabhängig von anderen Faktoren wie z.B. dem präoperativen Chemotherapieregime. Die Wahrscheinlichkeit für die Patienten mit höhergradigen Nebenwirkungen, eine histopathologische Komplettremission zu entwickeln, war mehr als dreimal höher als für Patienten ohne Toxizität (Odds-Ratio: 3,29, 95%-Konfidenzintervall: [1,01, 10,96]).Schlussfolgerung:Höhergradige akute Organtoxizität während präoperativer Radiochemotherapie bei Patienten mit multimodaler Therapie lokal fortgeschrittener Rektumkarzinome könnte einen frühen Prädiktor für das Ansprechen auf die Therapie in Bezug auf die histopathologische Remission darstellen. Ob dieser Parameter auch für die lokale Kontrolle sowie das Gesamtüberleben prädiktiv sein kann, wird in weiteren prospektiven Untersuchungen durch die Arbeitsgruppe der Autoren untersucht.