Impact of diet on EGFR-targeted treatment of colorectal cancer

Colorectal cancer (CRC) continues to be one of the most prevalent cancers and leading causes of cancer-related deaths in Western societies. Molecule-targeted therapies are being developed and tested in preclinical and clinical studies, with some promising results. One such class of therapeutics targets EGFR signaling, a pathway often deregulated in epithelial cancers. While these inhibitors have shown some effectiveness in clinical trials, the results have not been as promising and consistent as those reported in preclinical trials. The current study addresses these inconsistencies, expanding preclinical investigation to include multiple mouse models, genetic strains, and diet in an effort to better predict human response to these therapies. Two mouse models for CRC, ApcMin/+ and AOM, are used on distinct inbred strains as well as a shared F1 strain and are maintained on either a standard mouse chow (STD) or Western-style diet (WD). The efficacy of small molecule EGFR inhibitor AG1478 varies with respect to model, strain and diet. Additionally, human dietary supplementation is addressed, assessing the effect of supplementing a Western-style diet with high levels of calcium on AG1478 treatment of CRC. The two mouse models mentioned as well as an in vitro system including two human CRC cell lines, Caco-2 and HCT116, were used to evaluate the impact of low (0.05%) versus high (5%) calcium in vivo and increasing calcium concentrations in vitro (0.424 - 5.5mM) on AG1478-mediated tumor reduction. Calcium consistently impacts AG1478 with an additive effect on tumor growth reduction across each model system, however, with potentially toxic side effects in vivo. In addition, gender influences are revealed in response to diet and AG1478 inhibition of EGFR activity. We conclude that the diet consumed during cancer treatment, including the use of dietary supplements, may impact response to conventional therapies, and that understanding gene-environment-therapy interactions will be critical in developing and testing new therapies for CRC

Dietary calcium supplementation enhances efficacy but also toxicity of EGFR inhibitor therapy for colon cancer

The inverse correlation between levels of dietary calcium and colorectal cancer (CRC) incidence has been extensively investigated. However, the impact of supplemental calcium on cancer therapy remains unknown. We used four models of CRC, Caco-2 and HCT116 human cancer cell lines and ApcMin/+ and azoxymethane carcinogen-induced mouse models, to investigate the impact of a Western-style diet low in calcium (0.05%) vs. a similar diet but supplemented with calcium (5%) on therapeutic targeting of the epidermal growth factor receptor (EGFR). We found that calcium supplementation combined with pharmacologic blockade of EGFR results in an additive effect on tumor growth inhibition in all models. Unexpectedly, the combined use of dietary calcium supplementation and EGFR inhibitors also resulted in elevated toxicity suggesting that careful consideration be given when combining dietary supplements with prescribed cancer therapies

The histone methylase Set2p and the histone deacetylase Rpd3p repress meiotic recombination at the HIS4 meiotic recombination hotspot in Saccharomyces cerevisiae

The rate of meiotic recombination in the yeast Saccharomyces cerevisiae varies widely in different regions of the genome with some genes having very high levels of recombination (hotspots). A variety of experiments done in yeast suggest that hotspots are a feature of chromatin structure rather than a feature of primary DNA sequence. We examined the effects of mutating a variety of enzymes that affect chromatin structure on the recombination activity of the well-characterized HIS4 hotspot including the Set2p and Dot1p histone methylases, the Hda1p and Rpd3p histone deacetylases, the Sin4p global transcription regulator, and a deletion of one of the two copies of the genes encoding histone H3–H4. Loss of Set2p or Rpd3p substantially elevated HIS4 hotspot activity, and loss of Hda1p had a smaller stimulatory effect; none of the other alterations had a significant effect. The increase of HIS4 hotspot activity in set2 and rpd3 strains is likely to be related to the recent finding that histone H3 methylation by Set2p directs deacetylation of histones by Rpd3p

Efficacy of EGFR Inhibition Is Modulated by Model, Sex, Genetic Background and Diet: Implications for Preclinical Cancer Prevention and Therapy Trials

Molecule-targeted therapies are being widely developed and deployed, but they are frequently less effective in clinical trials than predicted based upon preclinical studies. Frequently, only a single model or genetic background is utilized using diets that are not relevant to that consumed by most cancer patients, which may contribute to the lack of predictability of many preclinical therapeutic studies. Inhibition of epidermal growth factor receptor (EGFR) in colorectal cancer was used to investigate potential causes for low predictive values of many preclinical studies. The efficacy of the small molecule EGFR inhibitor AG1478 was evaluated using two mouse models, ApcMin/+ and azoxymethane (AOM), both sexes on three genetic backgrounds, C57BL/6J (B6) and A/J (A) inbred strains and AB6F1 hybrids, and two diets, standard chow (STD) or Western-style diet (WD). AG1478 has significant anti-tumor activity in the B6-ApcMin/+ model with STD but only moderately on the WD and in the AOM model on an A background with a WD but not STD. On the F1 hybrid background AG1478 is effective in the ApcMin/+ model with either STD or WD, but has only moderate efficacy in the AOM model with either diet. Sex differences were also observed. Unexpectedly, the level of liver EGFR phosphorylation inhibition by AG1478 was not positively correlated with inhibition of tumor growth in the AOM model. Model-dependent interactions between genetic background and diet can dramatically impact preclinical results, and indicate that low predictive values of preclinical studies can be attributed to study designs that do not account for the heterogeneous patient population or the diets they consume. Better-designed preclinical studies should lead to more accurate predictions of therapeutic response in the clinic

Summary of pair-wise observations of AG1478 inhibitor effect on tumor growth.

<p>SD standard diet; WD, western diet; ↓ corrected p<0.05; trend, uncorrected p<0.05 but corrected p>0.05.</p

Western blot analysis of EGFR signal.

<p>Total EGFR and pEGFR in liver protein lysates from A) APC<i>^Min</i>^/+ and B) AOM mice, separately for each strain and sex. STD = standard diet, WD = western diet, “+”  =  AG1478 treatment and “−“  =  no AG1478 treatment. Below bar graphs are estimated effects and significance based on linear mixed models.</p

Effect of diet, sex and strain on AG1478-mediated tumor number reduction.

<p>A) <i>Apc^Min</i>^/+ small intestinal and colonic tumor number as a percentage of STD without AG1478 treatment for the B6 inbred and AB6F1 (shaded) strains; and B) AOM (A and AB6F1) colonic tumor number expressed as a percent of STD treatment. The raw values for average number of tumors/mouse are displayed below each graph. STD = standard diet, WD = western diet, “+”  =  AG1478 treatment and “−“  =  no AG1478 treatment. Standard Error of the Mean bars are shown for each treatment. Below bar graphs are estimated effects and significance based on linear mixed models.</p

Comparison of diet compositions.

<p>Comparison of diet compositions.</p

Effect of diet, sex and strain on AG1478-mediated tumor size(mm) reduction.

<p>A) <i>Apc^Min</i>^/+ small intestinal and colonic tumor size as a percentage of STD without AG1478 treatment for the B6 inbred and AB6F1 (shaded) strains; and B) AOM (A and AB6F1) colonic tumor size expressed as a percent of STD treatment. The raw values for average size of tumors/mouse are displayed below each graph. STD = standard diet, WD = western diet, “+”  =  AG1478 treatment and “−“  =  no AG1478 treatment. Standard Error of the Mean bars are shown for each treatment. Below bar graphs are estimated effects and significance based on linear mixed models.</p