Jeffrey T. Brasky

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 μm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.

Topical polyethylene glycol as a novel chemopreventive agent for oral cancer via targeting of epidermal growth factor response.

Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21cip1/waf1. In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

MicroRNAs as Novel Targets for NSAID Chemoprevention of Colon Carcinogenesis

Background & Objective: In the United States, colorectal cancer (CRC) is the third most prevalent and deadly malignancy. Development of novel CRC-specific abnormal DNA biomarkers may advance non-invasive, cost-efficient population-based CRC screening and ultimately reduces CRC death. DNA hypermethylation is a common epigenetic abnormality in CRCs and represents a promising class of cancer biomarkers. The objective of the current study was to develop optimal DNA hypermethylation-based biomarkers for use in fecesor serum-based average-risk CRC screening. Design: We first applied DNAmethylationmicroarray analysis in order to identify novel loci demonstrating neoplasia-specific methylation in the colon. This array analysis allowed us to investigate 55% of CpG islands within the genome and was applied to 17 primary CRCs relative to 8 non-neoplastic colonic tissues (NCs) from neoplasia-free subjects. The detected CRC-associated hypermethylation events were then individually measured in 113 colonic tissues comprising 51 CRCs, 9 adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs) using highly sensitive and quantitative real-time quantitative methylation-specific PCR (qMSP) assays. Receiver-operator characteristics (ROC) curve analysis was applied to the qMSP data in order to assess each individual event as well as combination of events for their ability to discriminate neoplastic from non-neoplastic cases. Results: Microarray-based global methylation profiles discriminated CRCs from NCs. A bioinformatic filtering of the microarray data identified 169 candidate CRC-associated hypermethylation events, including one previously validated hypermethylation marker for fecal DNA-based CRC detection, SFRP2. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from control NCs (p<.01). Of these ten events, methylation of VSX2 achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity; Area under ROC curve, or AUC, 0.93, p<1E-6). Similarly, CRC-NCs were significantly discriminated from control NCs by methylation of ALX3 (AUC 0.78, p<1E-4). The discrimination of CRC-NCs from control NCs was improved by a multi-locus methylation panel (AUC 0.83, p<1E-6) relative to ALX3. Although the sample numbers were small, two methylation events significantly distinguished adenomas from control NCs (p<.01). Conclusions: Systematic methylome analysis has identified 11 novel methylation events in neoplastic and non-neoplastic colonic mucosa from CRC patients that accurately discriminate CRC patients from controls. These markers merit further evaluation as candidate biomarkers for stool and circulating DNA-based CRC detection.

Mutual Regulation of Expression Between MUC4 and CDx2 in Colon Cancer Cells

Background:Mucins are a family of large glycoproteins that have been implicated in colorectal carcinogenesis. Our group has explored the role of MUC4, a transmembrane mucin, which is generally upregulated in most cancers (pancreas, lung etc), during colorectal carcinogenesis and noted that MUC4 is paradoxically downregulated in colorectal cancers. The progressive downregulation occurs since early stages of carcinogenesis (premalignant mucosa). However, the mechanism remains unclear. CDX2 is a caudal-related homeobox gene that has been known to be dysregulated in colon carcinogenesis. Intriguingly, a recent report implicated CDX2 in MUC4 regulation in Barretts esophagus. We, therefore, wanted to elucidate the role of CDX2 in the MUC4 loss in colon carcinogenesis Materials and Methods:HT29 cells were incubated in standard conditions and transiently transfected with CDX2 siRNA using Lipofectamine. Separately, HT29 cells were stably transfected withMUC4 shRNA and suitable control vector (CV). MUC4 shRNA transfected HT29 cells and CV transfected HT29 cells were treated with PI3K inhibitor LY294002. Protein lysates and RNA were obtained from the transfected cells and western blotting and qRT-PCR performed to assess the expression of MUC4, CDX2 and pAKT (S473). Results:In HT29 cells, a modest decrease in CDX2 expression by SiRNA (34% decreased CDX2 expression) concomitantly reduced MUC4 expression by 20%. Conversely, MUC4 shRNA transfection of HT29 cells (75% reduced MUC4 expression; p=0.05) resulted in 80% reduction in CDX2 mRNA expression and 60% reduction (p<0.02) in CDX2 protein levels. Since PI3K-AKT signaling cascade is known to modulate CDX2 levels, we assessed this pathway in MUC4 shRNA transfected HT29 cells, and demonstrated 50% increase (p<0.02) in Serine 473 phosphyrated AKT, which was mitigated by PI3K inhibitor, LY294002. However, despite the fact that MUC4 knockdown activated AKT, there was no effect of LY294002 treatment on the expression of CDX2 in MUC4 shRNA transfected HT29 cells, indicating that MUC4 modulates CDX2 expression via PI3K/pAKT independent manner. Conclusions: We report, herein, for the first time, that the dysregulation of two putative tumor suppressor genes, MUC4 and CDX2, is intimately related in colon cancer. Not only do CDX2 and MUC4 mirror each others expression but also mutually regulate each other. Further studies are ongoing to dissect this important novel pathway in colon carcinogenesis.

The Application of Metabolic Markers (GLUT1, GLUT4, TKTL1/2) to Formulate Novel Risk Stratification Strategies Against Colorectal Cancer

Background: Tumors ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). This nonoxidative conversion of glucose to lactate is activated by transketolase, an enzyme of the pentose phosphate pathway. For maintaining accelerated metabolism, tumors are known to have high requirements for glucose and thus have increased transport of glucose across the plasma membrane. Metabolic dependence of tumors on glucose utilization has been characterized by three genes; GLUT1, GLUT 4 and TKTL1/2. However, the expression patterns of the genes have not been explored fully for early stages of colorectal carcinogenesis. In this study we studied the expression pattern of these genes in colonic carcinogenesis (rat-AOM model and human CRC) and chemoprevention (by studying the effect of celecoxib in, Caco-2 and HCT-116 cell lines).Methods: Immunohistochemical expression of GLUT1, GLUT4, and TKTL1/2 was analyzed in human colon caner tissue array (consisting of 36 cases of colonic cancers and 12 cases each of normal, inflammatory and benign tumor tissues of the colon). In addition we analyzed 14 week AOM-treated rat colon tissue samples (early stage of colorectal carcinogenesis model) and saline-treated samples. Caco-2 and HCT-116 CRC cells were treated with 50μM celecoxib for 72hrs. Further analysis was carried out through real-time PCR using TKTL (1 & 2), GLUT (1 &4), PCNA and β-actin (control) Taqman probes. Results: Our results reveal an increased expression of GLUT1, GLUT4 and TKTL1/2 during CRC progression from adenoma to mucinous adenocarcinoma (Table 1). Increased expression of these genes was evident during early onset of carcinogenesis as shown in 14 week AOM-treated rat colon. Lastly, the cell culture data revealed that celecoxib caused a significant reduction in the expression of these metabolic markers in Caco-2 (p<.01) or HCT-116 (p<.05). These changes corresponded to the reduction in the proliferation marker PCNA in Caco-2 (95%) or in HCT-116 cells (39%) (Table 2). Conclusions: We, herein, demonstrate that expression of GLUT 1, GLUT4, and TKTL1/2 is significantly altered during early CRC and are key factors in cancer progression. Our studies show a significant increase in the glucose transporter 1 and 4, in relationship to cancer progression and early stages of colorectal carcinogenesis, thereby proposing potential biomarker role for these markers. TKTL1/2, on the other hand, seems to be up regulated in only a few of human colorectal tumors, which portends it to be a subset-specific biomarker. We further demonstrate that chemopreventive properties of celecoxib against colorectal cancer (Caco-2 and HCT-116 cells) may be modulated by the metabolic mediators involved in glycolysis. Further studies are needed to access the clinical application of these receptors as potential biomarkers for colorectal cancer. Table 1. Colon Tumor Tissue Array

Superoxide Dismutase 2 (Sod2) is Overexpressed at an Early Stage During Colorectal Carcinogenesis: A Putative Target for Celecoxib Chemoprevention

Background: The occurence of circulating epithelial cells and cell-free tumor DNA (cfDNA) in peripheral blood of patients suffering from progressive forms of cancers has first been reported already in 1960s. With the great acceleration of genetic diagnostics by methods of polymerase chain reaction (PCR) the field of non-invasive examination of circulating molecular cancer biomarkers has recently been re-discovered. Aims: To investigate occurence of cfDNA as potential marker of regional and distant metastatic progression of colorectal cancer and to trace the source of cfDNA in patients undergoing surgery treatment at different levels of radicality. Methods: In a group of 165 patients in various stages of the disease tissue samples were initially acquired either as biopsies or resections. Samples were tested for a presence of the most frequent somatic colorectal cancer mutations within KRAS, APC, TP53, BRAF and PIK3CA genes. In addition, multiple plasma samples (n=789) were acquired over a time period covering (i) initial examination, (ii) immediately preceding a surgery (iv) postsurgery and (v) subsequent follow-up. Cell-free tumor DNA was traced in patient plasma by targeting mutations previously detected in tumor tissue. Results: Among patients who were positive for at least one of the detected somatic mutations in tumor tissue (102/165, 62%), cfDNA was present in 39 pre-surgery plasma samples. The frequency of cfDNA was correlated to the disease stage with 0% in Stages 0 and I, 9% in Stage II, 29% in Stage III and 94% in Stage IV. All cfDNA-positive patients (n=21) who underwent radical resection (R0) were free of cfDNA in subsequent testing of samples several days following surgery. All but 4 patients undergoing non-radical surgery, such as partial hepatic resections or paliative surgical treatment, remained cfDNA-positive also after the surgery. In some of the R0 patients, the cfDNA has re-appeared after a period of 18 22 months as a result of disease progression though local nodes or newly discovered metastases with continuous monitoring of the remainder of the group still in progress. Conclusion: Examination of a presence of cfDNA based on scanning plasma samples for presence of specific tumor mutations is a suitable tool for non-invasive monitoring of the disease progression as well as evaluation of surgery outcome. Supported by Czech Ministry of Health grant no. NS9809.