Modular Cre/lox System and Genetic Therapeutics for Colorectal Cancer

The Cre/lox system is a powerful tool for targeting therapeutic effectors in a wide variety of human disorders. I review a Cre/lox Wnt-targeted system that has shown promise against Wnt-positive colorectal cancer cell lines. In addition to Wnt-specific targeting of cell death inducers, the modular nature of this gene therapy model system can be exploited by designing positive and negative feedback loops to either amplify or inhibit Wnt activity for experimental or therapeutic benefit. I discuss the structural components and performance parameters of the system, the implication of these findings with respect to cancer stem cells, as well as the general applicability of this system to any disorder characterized by differential gene expression. I also consider the issue of gene delivery as well as in vivo testing requirements necessary for the further characterization and development of this system

Extreme Fluctuations in Wnt/beta-Catenin Signaling as an Approach for Colon Cancer Prevention and Therapy

Abstract: Mutations in the Wnt/beta-catenin (Wnt) signaling pathway initiate most cases of sporadic colon cancers (CC); therefore one approach for CC prevention and therapy is to suppress Wnt activity. However, the prolonged suppression of the signaling pathway is detrimental to the intestinal stem cells that rely on Wnt signaling for survival. Furthermore, CC cells exposed for an extended period of time to a single Wnt signaling -modulating agent may develop resistance to it. A possible solution is suggested by our findings that: (a) butyrate, a colonic metabolite of dietary fiber, and synthetic histone deacetylase inhibitors that mimic the effects of butyrate, hyperactivate Wnt transcriptional activity in CC cells with mutations in the pathway, and (b) high fold changes of Wnt signaling induce high apoptotic levels in the mutated cells. Therefore, diet-and drug-based regimens that result in alternating periods of suppressed and hyper-activated Wnt signalling may become powerful anti-CC preventive and therapeutic approaches. The hypothesis can be tested by (1) evaluating the effects of drug-and dietbased regimens, which induce maximal fluctuations in Wnt signaling, on the adenoma burden of Apc/min mice, an established in vivo model of intestinal cancer, (2) epidemiological studies analyzing the dietary habits, such as daily fasting hours and fiber intake, of healthy individuals and patients with positive colonoscopies, and 352 D. L. Lazarova and M. Bordonaro (3) examining the ability of different diets to produce colonic lumen content with apoptotic and Wnt-modulatory functions

A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells

In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients

Crosstalk between Wnt Signaling and RNA Processing in Colorectal Cancer

<p>RNA processing involves a variety of processes affecting gene expression, including the removal of introns through RNA splicing, as well as 3' end processing (cleavage and polyadenylation). Alternative RNA processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. Deregulated Wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (CRC), has been linked to modified RNA processing, which may contribute to Wnt-mediated colonic carcinogenesis. Crosstalk between Wnt signaling and alternative RNA splicing with relevance to CRC includes effects on the expression of Rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative RNA splicing that is influenced by Wnt activity. In addition, Tcf4, a crucial component of Wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. Modulation of 3' end formation, including of the Wnt target gene <i>COX-2</i>, also can influence the neoplastic process, with implications for CRC. While many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron-independent gene expression. Effects of Wnt activity on the RNA metabolism of the intronless Wnt-target gene <i>c-jun</i> is a likely contributor to cancer development. Further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates Wnt activity in CRC cells, and also modulates RNA processing; therefore, the interplay between Wnt activity, the modulation of this activity by butyrate, and differential RNA metabolism in colonic cells can significantly influence tumorigenesis. Determining the role played by altered RNA processing in Wnt-mediated neoplasia may lead to novel interventions aimed at restoring normal RNA metabolism for therapeutic benefit. Therefore, this minireview presents a brief overview of several aspects of RNA processing of relevance to cancer, which potentially influence, or are influenced by, Wnt signaling activity.</p

The processing and accumulation of RNA from intron-dependent and intron-independent genes

This study is concerned with how sequences from the intron-containing mouse $\beta$-globin (M$\beta$G) gene and the naturally intronless herpes simplex virus-1 thymidine kinase (TK) gene influence the processing and accumulation of mRNA. RNase mapping was used to assay total RNA from transiently transfected COS-1 cells. The M$\beta$G gene was shown to require at least one functional intron for mRNA accumulation. Deletion of intron 2 alone depressed M$\beta$G transcript accumulation more than deletion of intron 1 alone; this is due to both differences in the introns themselves, and to the spacing between the introns and two elements: a required exonic element (EE) and the M$\beta$G polyadenylation site. Also, an unpaired 5\sp\prime splice-site can depress M$\beta$G mRNA accumulation even in the presence of the EE. M$\beta$G-TK chimeras were analyzed to study how intron-independent gene TK sequences influence M$\beta$G mRNA accumulation. Insertion of a 1.1kb TK gene fragment partially ameliorated the mRNA depletion seen with intronless, but not 3$\prime$ splice-site deleted, M$\beta$G transcripts. This demonstrated that the mechanism of mRNA depletion differs between these deletions. In addition, the ability of TK sequence to restore M$\beta$G accumulation was highly dependent on its position relative to the 5\sp\prime end of the chimera. This is consistent with a TK sequence-dependent co-transcriptional channelling of transcripts into an intron-independent metabolic pathway. A polyA trap was used to answer the question of whether the low accumulation of transcripts from intronless M$\beta$G genes are due to an inhibition of 3\sp\prime end processing. Data from two different sets of polyA trap constructs are most consistent with M$\beta$G 3\sp\prime end processing taking place despite the absence of introns. Therefore, the low accumulation of transcripts from intronless M$\beta$G genes is not primarily due to a lack of 3\sp\prime end processing