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 smallmolecule 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 mM 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.

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 paraffinembedded 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 p16 INK4a 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 p16 INK4a expression (p 5 0.001). Patients with HPV16 DNA load median and low p16 INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p 5 0.023, multivariate p 5 0.042; p16 INK4a : univariate p 5 0.021), and OS (HPV16 DNA load: univariate p 5 0.02, multivariate p 5 0.03). Moreover, a combined HPV16 DNA load and p16 INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p 5 0.019, p 5 0.04 and p 5 0.031). In conclusion, these data indicate that HPV16 DNA load and p16 INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.The majority of anal squamous cell carcinoma (SCC) is considered to originate from infection with human papilloma viruses (HPV). 1,2 Especially the role of high-risk oncogenic HPV types, most prominently HPV type 16 (HPV16), in the etiology of anal SCC is well established by a multitude of epidemiological and experimental investigations. 3,4 As a consequence, technologies for the detection of HPV DNA may constitute valuable screening tools for anal malignancies and are of important clinical relevance, especially after the development of a preventive vaccine for both men and women. 5 Depending on the method used, the prevalence of HPV DNA in anal carcinoma ranges from 75% to 100% with HPV16 (>75%) and less frequent HPV18 (<10%) detection in the majority of cases. 6,7 In clinical series of head and neck as well as cervical SCC, recent data indicate that treatment response to radiotherapy (RT) or chemoradiotherapy (CRT) is superior in HPV-positive tumors as compared with their HPV-negative counterparts. [8][9][10] An increased sensitivity of HPV-positive tumor cells towards irradiation and chemotherapeutic agents was also observed in vitro and in animal studies. [11][12][13] However, as the detection rate of HPV DNA in anal SCC commonly exceeds 90%, a comparison between sole HPV-positive and HPV-negative cancers with respect to treatment respon...

We examined the prognostic role of immune markers programmed cell death protein-1 (PD-1) and its ligand (PD-L1), CD8 tumor-infiltrating lymphocytes (TILs), FOXP3+ Tregs and phosphorylated Caspase-8 (T273) in patients with anal squamous cell cancer (ASCC) treated with standard chemoradiotherapy (CRT). The baseline immunohistochemical expression of immune markers was correlated with clinicopathologic characteristics, and cumulative incidence of local failure, disease-free survival (DFS) and overall survival (OS) in 150 patients, also in the context of human papilloma virus 16 (HPV16) DNA load and p16 expression. After a median follow-up of 40 mo (1-205 mo), the 5-y cumulative incidence of local failure and DFS was 19.4% and 67.2%, respectively. Strong immune marker expression was significantly more common in tumors with high HPV16 viral load. In multivariant analysis, high CD8 and PD-1+ TILs expression predicted for improved local control ( = 0.023 and = 0.007, respectively) and DFS ( = 0.020 and = 0.014, respectively). Also, high p16 ( = 0.011) and PD-L1 ( = 0.033) expression predicted for better local control, whereas high FOXP3+ Tregs ( = 0.050) and phosphorylated Caspase-8 ( = 0.031) expression correlated with superior DFS. Female sex and high HPV16 viral load correlated with favorable outcome for all three clinical endpoints. The present data provide, for the first time, robust explanation for the favorable clinical outcome of HPV16-positive ASCC patients harboring strong immune cell infiltration. Our findings are relevant for treatment stratification with immune PD-1/PD-L1 checkpoint inhibitors to complement CRT and should be explored in a clinical trial.

Activation of Wnt/β-catenin signaling plays a central role in the development and progression of colorectal cancer (CRC). The Wnt-transcription factor, TCF7L2, is overexpressed in primary rectal cancers that are resistant to chemoradiotherapy (CRT) and TCF7L2 mediates resistance to CRT. However, it is unclear whether the resistance is mediated by a TCF7L2 inherent mechanism or Wnt/β-catenin signaling in general. Here, inhibition of β-catenin by siRNAs or a small molecule inhibitor (XAV-939) resulted in sensitization of CRC cells to CRT. To investigate the potential role of Wnt/β-catenin signaling in controlling therapeutic responsiveness, non-tumorigenic RPE-1 cells were stimulated with Wnt-3a, a physiological ligand of Frizzled-receptors, which increased resistance to CRT. This effect could be recapitulated by overexpression of a degradation-resistant mutant of β-catenin (S33Y), also boosting resistance of RPE-1 cells to CRT, which was, conversely, abrogated by siRNA-mediated silencing of β-catenin. Consistent with these findings, higher expression levels of active β-catenin were observed as well as increased TCF/LEF reporter activity in SW1463 cells that evolved radiation resistance due to repeated radiation treatment. Global gene expression profiling identified several altered pathways, including PPAR signaling and other metabolic pathways, associated with cellular response to radiation. In summary, aberrant activation of Wnt/β-catenin signaling not only regulates the development and progression of colorectal cancer, but also mediates resistance of rectal cancers to chemoradiotherapy. Implications Targeting Wnt/β-catenin signaling or one of the downstream pathways represents a promising strategy to increase response to CRT.

Liver damage is a serious clinical complication of ␥-irradiation. We therefore exposed rats to singledose ␥-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-␥ 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 ␥-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.

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.

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.

x-Irradiation of the liver induced changes of hepcidin gene expression that are probably induced by acute phase mediators produced within the liver itself.