Abstract: BACKGROUND.: The Akt/mammalian target of the rapamycin (mTOR) signaling pathway represents a promising target for cancer therapy. The phosphorylation status of Akt and of mTOR's phosphorylation target eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is often used to assess the activity of Akt and mTOR signaling. The purpose of this study was to determine whether primary tumors differ from their metastasis in their expression of pAkt and p4E-BP1. METHODS.: Primary breast tumors and their distant metastases surgically resected from the same patients were evaluated with immunohistochemical analysis (IHC) for pAkt (Ser473) and p4E-BP1 (Ser65). The agreement between the IHC results for the primary tumor and metastases was evaluated with Cohen kappa (kappa). RESULTS.: Most primary breast tumors and metastatic tumors expressed pAkt (76% of each). Of the 23 matched evaluable pairs, however, 11 (47.8%) had discordant IHC results (kappa -0.31; 95% confidence interval [CI], -0.49 to -0.13). Similarly, although most of the primary and metastatic tumors were positive for p4E-BP1 (75% and 74%), of the 23 matched evaluable pairs, 8 (47.8%) were discordant (kappa 0.10; 95% CI, -0.33-0.52). CONCLUSIONS.: In this series, most primary breast tumors and metastases expressed pAkt and p4E-BP1 by IHC. Concordance between IHC findings in primary tumors and metastases was poor, however. Further work is needed to determine whether this reflects true biological heterogeneity or poor reproducibility of IHC with phosphospecific antibodies, and to identify which biomarkers can be assessed most reproducibly in primary tumors to predict activity of Akt/mTOR signaling and sensitivity to pathway inhibitors. Cancer 2008. (c)2008 American Cancer Society.
Abstract: INTRODUCTION: Although most vaccines target foreign infectious agents, therapeutic cancer vaccines target both well-established and metastatic tumor cells expressing tumor antigens. Active immunotherapy is intended to enhance or activate the immunosurveillance of an individual through a therapeutic vaccine. Renal cell carcinoma (RCC) is one of the most immunoresponsive cancers in humans, which in turn makes it an ideal candidate for immune based therapies. METHOD: Several types of therapeutic vaccines have been tested and applied in the clinical setting and can be divided into cell-based vaccines including direct application of inactivated autologous tumor cells, gene modified tumor cell-based, dendritic cell-based (expressing RCC derived tumor antigens), and non-cell-based vaccines. This review will examine the current status of cell-based vaccine immunotherapy and focuses on non-cell-based vaccine strategies. CONCLUSION: Recent advances in molecular targeting therapy have introduced a battery receptor tyrosine kinase (RTK) and mTOR inhibitors that provide promising treatment options, however, the tolerability of tumor vaccines and the success of clinical effectiveness in selected populations combined with recent advances in cellular therapies warrant the continued exploration of novel methods of tumor vaccine therapies in the clinical setting.
Abstract: BACKGROUND: The authors recently reported that transgenic mice (hGAS) expressing pharmacologic levels of progastrin (PG) (> 10 nM to 100 nM) exhibited increased susceptibility to colon carcinogenesis in response to azoxymethane (AOM). It is not known whether PG functions as a cocarcinogen at the concentrations observed in patients with hypergastrinemia (approximately 1.0 nM). METHODS: The authors generated transgenic mice that overexpressed either wild-type (wtPG) or mutant (mtPG) human PG in the intestinal mucosa using the murine fatty acid binding protein (Fabp) promoter. Fabp-PG mice and their wild-type littermates were treated with AOM, and their colons were examined for preneoplastic (aberrant crypt foci [ACF]) and neoplastic (adenomas [Ads] and adenocarcinomas [AdCas]) lesions after 2 weeks and 6 months of treatment. RESULTS: ACF and tumors were significantly more common (by a factor of approximately 2) in colon specimens from both Fabp-wtPG mice and Fabp-mtPG mice relative to wild-type mice. It is noteworthy that the multiplicity of ACF and the total number of small and large Ads and AdCas were significantly greater in colon specimens from Fabp-PG mice compared with colon specimens from wild-type mice, irrespective of gender. CONCLUSIONS: The results of the current study suggest that at concentrations (approximately 1.0 nM) far lower than the ones observed in hGAS mice, PG functions as an equally potent cocarcinogen and significantly increases the risk of colon carcinogenesis in response to AOM. Thus, PG may represent a clinically relevant target molecule in patients with hypergastrinemia or colon carcinoma.
Abstract: KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T-->C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.
Abstract: Aberrant crypt foci (ACFs) may be the earliest recognizable histologic precursor lesion for colon cancer. ACF may develop from a complex of events, including the development of cryptal hyperproliferation, defects in the rate of apoptosis, and abnormalities in cellular adhesion. In this study, we hypothesized that human ACF would exhibit discrete differences in cell adhesion proteins compared with normal mucosa of biologic markers associated with colon cancer. ACFs were isolated from resected colon mucosa from 45 patients undergoing surgery for colon cancer. We evaluated the protein expression of 3 biologic markers that may be related to the progression of aberrant crypt foci to tumors: carcinoembryonic antigen, E-cadherin, and sialyl Tn antigen. In general, ACFs located near cancers in the right colon were more often hyperplastic than dysplastic; this was more noticeable in the left colon. Carcinoembryonic antigen expression was found to be more intense in apical portions of ACF crypts, with sialyl Tn antigen moderately increased, whereas E-cadherin diffusely stained throughout crypts within ACFs. There are significant biologic changes in potential tumor markers that accompany the early transformation of the normal glandular epithelium, some of which are expressed very early in the colon at the stage of appearance of ACF.
Abstract: Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.
Abstract: BACKGROUND & AIMS: We recently reported that transgenic mice overexpressing progastrin were at a higher risk for developing colon cancers in response to azoxymethane (AOM), whereas mice overexpressing gastrin-17 were at a reduced risk. To examine further the role of gastrins in colon carcinogenesis, we generated gastrin gene knockout mice (GAS-KO). METHODS: The height and proliferative index (PI) of colonic crypts were similar in GAS-KO and wild-type (WT) mice, suggesting that the absence of gastrins in GAS-KO mice did not significantly affect the growth of colonic mucosa. GAS-KO and WT mice were treated with AOM for 3-4 weeks; control mice received saline. RESULTS: Colonic proliferation in response to AOM was significantly increased in GAS-KO vs. WT mice. Aberrant crypt foci (ACFs) were similarly increased significantly by approximately 2-5-fold in GAS-KO vs. WT mice after 2 weeks of AOM treatment. Female GAS-KO mice developed adenomas (Ads) and adenocarcinomas (AdCAs) at earlier times ( approximately 10 months) than the male GAS-KO mice and the male and female WT mice ( approximately 12 months). The total numbers of Ads and AdCAs were significantly higher in GAS-KO than in WT mice. CONCLUSIONS: These results suggest the novel possibility that loss of gastrin expression (and hence amidated gastrins) significantly increases susceptibility to colon carcinogenesis in response to AOM. Previous studies with FVB/N transgenic mice similarly suggested a protective role of amidated gastrins against colon carcinogenesis, which supports the present findings of an increase in colon carcinogenesis in GAS-KO mice lacking normal physiological levels of amidated gastrins.
Abstract: Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.
Abstract: BACKGROUND & AIMS: Processing intermediates of preprogastrin (gly-gastrin and progastrin), termed nonamidated gastrins, are mitogenic for several cell types including colonic epithelial cells. However, presently it is not known if nonamidated gastrins play a role in colon carcinogenesis and if the effects are similar to those of amidated gastrins. METHODS: Colon carcinogenesis in response to azoxymethane (AOM) was examined in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS), compared with that in wild-type (WT) mice. RESULTS: In AOM-treated groups, the total number of tumors per colon was significantly higher in hGAS (4.8+/-0.34) than INS-GAS (3.0+/-0.16) and WT (2.7+/-0.35) mice. Total numbers of adenocarcinomas and adenomas per animal colon were also significantly higher in hGAS than INS-GAS and WT mice. The size of the tumors was greater in hGAS mice, resulting in a significantly higher tumor burden per mouse in the hGAS mice than INS-GAS and WT mice. Although >90% of the tumors were located in the distal half of the colon in INS-GAS and WT mice, a significant number (42%) were present at the proximal end of the colon in hGAS mice. CONCLUSIONS: The results suggest that the risk for developing colon carcinomas and adenomas in response to AOM is significantly increased in mice expressing high levels of progastrin, but not amidated gastrins.
Abstract: Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.
Abstract: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine carcinogen in the human diet and is a colon carcinogen in the rat. N-Acetyltransferase-2 (NAT2) catalyzes the conversion of PhIP and other heterocyclic amines to a DNA-reactive form. NAT2 has a polymorphic distribution in humans and other mammals, including rats. The rapid NAT2 genotype has been shown to be associated with increased colorectal cancer risk in some, but not all, human epidemiological studies. This investigation was designed to study the role of acetylator genotype in PhIP-induced colon carcinogenesis using aberrant crypt foci (ACF) as an intermediate biomarker. Five-week-old male, rapid-acetylator Fischer 344 (F344) rats and slow-acetylator Wistar-Kyoto (WKY) rats were fed the semipurified AIN76A diet with 0.01% PhIP, 0.04% PhIP, or no PhIP (control) for 8 weeks. PhIP induced ACF in both rapid- and slow-acetylator rats; 0.04% PhIP induced more ACF than 0.01% PhIP. There was no difference in the number of ACF between rapid- and slow-acetylator rats that were fed 0.01% PhIP. However, 0.04% PhIP induced 2-fold higher ACF and a greater dose-dependent increase in PhIP-induced ACF in the rapid-acetylator F344 rats compared with the slow-acetylator WKY rats. The results support human epidemiological studies showing higher risk for colorectal cancer in rapid acetylators who frequently consume meat that is very well done.
Abstract: We assessed the effects of 78 potential chemopreventive agents in the F344 rat using two assays in which the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon was the measure of efficacy. In both assays ACF were induced by the carcinogen azoxymethane (AOM) in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last AOM injection, animals were evaluated for the number of aberrant crypts detected in methylene blue stained whole mounts of rat colon. In the initiation phase protocol agents were given during the period of AOM administration, whereas in the post-initiation assay the chemopreventive agent was introduced during the last 4 weeks of an 8 week assay, a time when ACF had progressed to multiple crypt clusters. The agents were derived from a priority listing based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. During the initiation phase carboxyl amidoimidazole, p-chlorphenylacetate, chlorpheniramine maleate, D609, diclofenac, etoperidone, eicosatetraynoic acid, farnesol, ferulic acid, lycopene, meclizine, methionine, phenylhexylisothiocyanate, phenylbutyrate, piroxicam, 9-cis-retinoic acid, S-allylcysteine, taurine, tetracycline and verapamil were strong inhibitors of ACF. During the post-initiation phase aspirin, calcium glucarate, ketoprofen, piroxicam, 9-cis-retinoic acid, retinol and rutin inhibited the outgrowth of ACF into multiple crypt clusters. Based on these data, certain phytochemicals, antihistamines, non-steroidal anti-inflammatory drugs and retinoids show unique preclinical promise for chemoprevention of colon cancer, with the latter two drug classes particularly effective in the post-initiation phase of carcinogenesis.
Abstract: We assessed the effects of 41 potential chemopreventive agents in the F344 rat using the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon as the measure of efficacy. ACF were induced by the carcinogen azoxymethane in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last azoxymethane injection, animals were evaluated for the number of aberrant crypts detected in methylene blue-stained whole mounts of rat colon. The 41 agents were derived from a priority listing that was based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. The list of agents included representative examples of phytochemicals, vitamins, minerals, inhibitors of proliferation, inducers of Phase 1 and Phase 2 metabolism systems, nonsteroidal anti-inflammatory agents, and differentiation agents. Eighteen agents were positive in the assay, significantly reducing the incidence of ACF at least in one of two doses tested. As a chemical class, the nonsteroidal anti-inflammatory drugs, which included ibuprofen, ketoprofen, piroxicam, and indomethacin, were most active; other less potent agents were arginine, butylated hydroxyanisole, curcumin, diallyl sulfide, difluoromethylornithine, 18 beta-glycyrrhetinic acid, indole-3-carbinol, oltipraz, purpurin, rutin, and the sodium salts of butyrate, selenite, and thiosulfate. Twenty-three agents did not inhibit ACF; included among these were several agents that promoted the development of ACF at one or both doses tested: benzyl isothiocyanate,calcium glucarate, catechin, dihydroepiandosterone, fluocinolone acetonide,folic acid, levamisole, 2-mercaptoethanesulfonic acid, nordihydroguiaretic acid, potassium glucarate, propyl gallate, beta-sitosterol, sodium cromolyn, sodium molybdate, and sulfasalazine. The aberrant crypt assay demonstrates reasonable specificity and sensitivity in predicting which agents are likely to prevent colon cancer.
Abstract: Aberrant crypts are aggregates of single to multiple colonic crypts evidencing hallmarks of dysplasia and may be the earliest detectable pathological lesions for colon cancer. The aberrant crypt assay has been developed in 2 protocols. In one, putative chemoprevention agents are tested for inhibitory effects when administered concomitantly with a carcinogen. In the other, the objective of this study, aberrant crypts were induced in F344 rats by parenteral injection of the colon carcinogen azoxymethane (AOM) and allowed to develop for 4 weeks, when an average of 90-100 aberrant crypt foci per colon were found in the methylene blue-stained colon. Then, during the second 4 weeks of the experiment, aberrant crypts were allowed to further develop to a frequency of > 150 foci per colon, a time when multi-crypt foci were observed. During this time we tested the inhibitory effects of 4 analgesic drugs and 2 differentiation agents for effects of aberrant crypt growth and development. We found the non-steroidal anti-inflammatory drugs piroxicam, aspirin and ibuprofen, but not acetaminophen, to be effective in suppressing aberrant crypt formation or the progression to foci of multiple aberrant crypts. Treatment with chemosuppressing agents 13-cis-retinoic acid (13-cRA) and 4-hydroxyphenretinamide (4-HPR), known differentiating agents, however, did suppress expansion of aberrant crypt foci, with 13-cRA being the much more potent agent.
Abstract: Micronuclei frequency, a marker of genotoxicity, was studied within a trial of alpha-tocopherol for chemoprevention of oral leukoplakia. Oral swabs were obtained from two sites, the leukoplakia lesion and normal-appearing mucosa, at baseline and following 24 weeks of therapy with 400 international units of alpha-tocopherol twice daily. These specimens were analyzed for micronuclei frequency. The major risk factors for oral carcinogenesis in the group studied were cigarette smoking and alcohol consumption. alpha-tocopherol therapy produced a significant reduction in micronuclei frequencies in specimens from both the visible lesions (P < 0.01) and the normal-appearing mucosa (P < 0.01). The micronuclei frequencies, both at baseline and following therapy, were greater in specimens taken from the lesion than in those from the normal-appearing mucosa. Although these results indicate that alpha-tocopherol has a beneficial effect in oral carcinogenesis, there was no significant clinical or histological response associated with the change in micronuclei frequency. Micronuclei frequency has not yet been validated as a biomarker for cancer incidence, and consequently, its utility as an intermediate end point for chemoprevention trials is not known. Determining clinical significance of micronuclei frequency patterns in oral carcinogenesis and chemoprevention will require further study.
Abstract: Biomarkers are being sought that could serve as surrogate end points for chemoprevention trials. Micronuclei, cytoplasmic fragments of DNA, have been proposed as a biomarker and studied in oral pre-malignancy. This study evaluated micronuclei frequency in a randomized chemoprevention trial of oral pre-malignancy. A recent clinical trial evaluated the responses of pre-malignant oral lesions to 3 months of therapy with isotretinoin followed by 9 months of either low-dose isotretinoin or beta-carotene. For 57 study participants, micronuclei were counted in mucosal scrapings of the lesion and in normal-appearing mucosa at baseline and following 3 months and 12 months of therapy. Micronuclei counts were higher in scrapings from the lesion than in the normal-appearing mucosa. Following 3 months of isotretinoin, the micronuclei counts in scrapings of the lesion were significantly reduced. With treatment, the mean micronuclei count declined at 3 months. In a randomized comparison, both isotretinoin and beta-carotene maintained the suppression of micronuclei. The change in micronuclei count was not associated with the clinical or histological response to treatment. Chemoprevention treatment with isotretinoin led to a reduction in frequency of micronuclei, a marker of recent DNA injury, which was then maintained by both isotretinoin and beta-carotene.
Abstract: Bronchial micronuclei, small fragments of extra-nuclear DNA formed during cell division, provide a non-specific but quantifiable marker of DNA damage. Micronuclei have been used to assess carcinogen exposure and as an intermediate endpoint in chemoprevention trials. As part of an ongoing chemoprevention trial, heavy smokers underwent screening bronchoscopy, with biopsies taken at 6 standardized sites. Micronuclei counts were obtained for each site in each of the 40 volunteers found to have squamous metaplasia. Unlike squamous metaplasia, the average micronuclei counts among these heavy smokers were not associated with smoking history. Micronuclei counts were also not associated with the presence or extent of metaplasia. A striking degree of intra-individual variability was observed by comparing the micronuclei counts from different biopsy sites within individuals. The findings suggest that use of micronuclei from single sites may be misleading as a marker of carcinogen exposure or as an estimate of cancer risk. Serial measurements in individuals may provide the most useful information concerning carcinogenic exposure and the impact of chemopreventive agents.
