Purpose: Voltage-gated Kv potassium channels, like ether a go-go (EAG) channels, have been recognized for their oncogenic potential in breast cancer and other malignant tumors. Experimental Design: We examined the molecular and functional expression of Kv channels in human colonic cancers and colon of mice treated with the chemical carcinogens dimethylhydrazine and N-methyl-N-nitrosourea. The data were compared with results from control mice and animals with chemically induced DSS colitis. Results: Electrogenic salt transport by amiloride-sensitive Na + channels and cyclic AMPâ ctivated cystic fibrosis transmembrane conductance regulator Cl À channels were attenuated during tumor development and colitis, whereas Ca 2+

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.

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.

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.

Although known for more than 20 years, the molecular identity of epithelial Ca(2+)-activated Cl(-) channels remains obscure. Previous candidate proteins did not hold initial promises, and thus, new hope is put into the recently identified family of bestrophin proteins, as they reflect many of the properties found for native channels. Mutations in the bestrophin gene cause an autosomal form of macular dystrophy of the retina. Bestrophin 1 is assumed to form the basolateral Ca(2+)-activated Cl(-) channel in the retinal pigment epithelium of the eye. Other data suggest that bestrophin is a regulator of voltage gated Ca(2+) channels. Structural information on bestrophins is available and a Cl(-) selective filter has been localized to the second transmembrane domain of bestrophin. It is possible that bestrophins function as physiologically regulated Cl(-) channels only in association with additional subunits and auxiliary proteins. Little is known about expression of bestrophin in gland acinar cells, which show a pronounced Ca(2+)-activated Cl(-) secretion. In airways and intestinal epithelia, bestrophins could be particularly important in diseases such as cystic fibrosis and secretory diarrhea.

Plasma membrane potassium (K+) channels are required for cell proliferation. Evidence is growing that K+ channels play a central role in the development and growth of human cancer. Here we examine the contribution and the mechanism by which K+ channels control proliferation of T84 human colonic carcinoma cells. Numerous K+ channels are expressed in T84 cells, but only voltage-gated K+ (Kv) channels influenced proliferation. A number of Kv channel inhibitors reduced DNA synthesis and cell number, without exerting apoptotic or toxic effects. Expression of several Kv channels, such as EagI, Kv 3.4 and Kv 1.5, was detected in patch clamp experiments and in fluorescence-based assays using a voltage sensitive dye. The contribution of EagI channels to proliferation was confirmed by siRNA, which abolished EagI activity and inhibited cell growth. Inhibition of Kv channels did not interfere with the ability of T84 cells to regulate their cell vol, but it restricted intracellular pH regulation. In addition, inhibitors of Kv channels, as well as siRNA for EagI, attenuated intracellular Ca2+ signaling. The data suggest that Kv channels control proliferation of colonic cancer cells by affecting intracellular pH and Ca2+ signaling.

Chemoradiotherapy (CRT) represents a standard treatment for many human cancers, frequently combined with radical surgical resection. However, a considerable percentage of primary cancers are at least partially resistant to CRT, which represents a substantial clinical problem, because it exposes cancer patients to the potential side effects of both irradiation and chemotherapy. It is therefore exceedingly important to determine the molecular characteristics underlying CRT-resistance and to identify novel molecular targets that can be manipulated to re-sensitize resistant tumors to CRT. In this review, we highlight much of the recent evidence suggesting that the signal transducer and activator of transcription 3 (STAT3) plays a prominent role in mediating CRT-resistance, and we outline why inhibition of STAT3 holds great promise for future multimodal treatment concepts in oncology.

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.