Yolanda Stypula

Role of Cytoskeleton in Controlling the Disorder Strength of Cellular Nanoscale Architecture

Cytoskeleton is ubiquitous throughout the cell and is involved in important cellular processes such as cellular transport, signal transduction, gene transcription, cell-division, etc. Partial wave spectroscopic microscopy is a novel optical technique that measures the statistical properties of cell nanoscale organization in terms of the disorder strength. It has been found previously that the increase in the disorder strength of cell nanoarchitecture is one of the earliest events in carcinogenesis. In this study, we investigate the cellular components responsible for the differential disorder strength between two morphologically (and hence microscopically) similar but genetically altered human colon cancer cell lines, HT29 cells and Csk shRNA-transfected HT29 cells that exhibit different degrees of neoplastic aggressiveness. To understand the role of cytoskeleton in nanoarchitectural alterations, we performed selective drug treatment on the specific cytoskeletal components of these cell types and studied the effects of cytoskeletal organization on disorder strength differences. We report that altering the cell nanoarchitecture by disrupting cytoskeletal organization leads to the attenuation of the disorder strength differences between microscopically indistinguishable HT29 and CSK constructs. We therefore demonstrate that cytoskeleton plays a role in the control of cellular nanoscale disorder.

Dysregulation of microRNAs in colonic field carcinogenesis: implications for screening.

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.

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.

Biological mechanisms underlying structural changes induced by colorectal field carcinogenesis measured with low-coherence enhanced backscattering (LEBS) spectroscopy.

We previously reported the utility of Low-Coherence Enhanced Backscattering (LEBS) Spectroscopy in detecting optical changes in uninvolved rectal mucosa, changes that are indicative of the presence of advanced colorectal adenomas elsewhere in the colon (field carcinogenesis). We hypothesized that the alterations in optical signatures are due to structural changes in colonocytes. To elucidate those colonocyte changes, we used LEBS and an early time point in an animal model of colorectal field carcinogenesis – rats treated with azoxymethane (AOM). Changes in LEBS markers in intact mucosa from AOM-treated rats could be at least partially attributed to changes in colonocytes. To investigate the molecular mechanisms underlying the colonocyte abnormalities in premalignant colon, we took a candidate approach. We compared expression profiles of genes implicated directly or indirectly in cytoskeletal dysregulation in colorectal tissues from saline-treated versus AOM-treated rats. Our data suggest that a number of genes known to affect colon tumorigenesis are up-regulated in colonocytes, and genes previously reported to be tumor suppressors in metastatic cancer are down-regulated in colonocytes, despite the colonocytes being histologically normal. To further understand the role of the cytoskeleton in generating changes in optical markers of cells, we used pharmacological disruption (using colchicine) of the cytoskeleton. We found that differences in optical markers (between AOM- and control-treated rats) were negated by the disruption, suggesting cytoskeletal involvement in the optical changes. These studies provide significant insights into the micro-architectural alterations in early colon carcinogenesis, and may enable optimization of both bio-photonic and molecular risk stratification techniques to personalize colorectal cancer screening.

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.

Abstract A25: A novel spectroscopic technology to image the native chromatin nanostructure in live cells

Proper regulation of higher-order chromatin structure is essential for normal gene regulation and cellular function. We have previously found that the nanoscale chromatin structure is significantly altered in early and field carcinogenesis using novel spectroscopic methods in parallel with biological assays (Backman and Roy, J Cancer, 2013, 3:251-261; Subramanian et al, Cancer Res, 2009, 13:5357-63; Stypula-Cyrus et al, PLoS One, 2013, 5:e64600). This was done in fixed human and animal model samples, suggesting that genetic/epigenetic alterations can serve as the earliest marker for neoplastic transformation. While chromatin is well understood at the nucleosomal level ( 200nm), little is known about the higher-order chromatin structure between these length scales. Current techniques available to study cellular structures below the diffraction limit ( The BaSIS instrument was built into a commercial inverted microscope (Leica DMIRB) equipped with a high NA oil immersion objective with broadband illumination provided by a Xenon lamp. Refractive index fluctuations are measured by sampling backscattered light at each wavelength 500-700nm using a combination of a liquid crystal tunable filter (LCTF) and a CMOS camera. HeLa and CHO cells were first imaged in petri dishes with coverslip bottoms, and then incubated with Hoechst 33342, a nuclear stain that binds to AT-rich regions of the genome and has been reported to cause double-stranded breaks (DSBs) in the DNA (Pfeiffer et al, Mutagenesis, 2000, 4: 289-302). Additionally, mock-staining experiments were performed to compare the changes in nuclear structure due to Hoechst 33342 excitation compared to UV light exposure alone. We utilized a γ-H2A.X-Alexa488 conjugated antibody after Hoechst- and mock-staining to compare observed changes in BaSIS signal with the formation of DSBs. Using BaSIS, we show for the first time that the excitation of Hoechst 33342 immediately alters the native nuclear nanostructure and induces formation of DSBs, confirmed by the rapid phosphorylation of H2A.X. In our mock-stained control, we observed an average increase of 0.006% and 0.001% signal after UV exposure (p-value > 0.5), whereas the stained cells display a 17.01% and 7.1% decrease in HeLa and CHO nuclei, respectively (p-value In conclusion, BaSIS is a powerful tool for studying the dynamics of chromatin nanostructure and can serve as a natural supplement to super-resolution fluorescence techniques, providing quantified information about native cellular organization. With this technique, we demonstrated that using the Hoechst DNA-binding dye causes irreversible alterations in chromatin structure at time-scales (seconds) not previously recognized. As a result, BASIS can be applied to a broad range of critical studies in chromatin research. Current and future research include: (i) mRNA transport and the accessibility of euchromatin and heterochromatin to transcription factors; (ii) why and how high-order chromatin structure changes in cancer progression; (iii) the role of nuclear architecture as an epigenetic regulator of gene expression; and (iv) the effect of metabolism on chromatin structure; (v) damage/repair mechanisms and potentially, chemotherapeutic efficacy. Citation Format: Yolanda Stypula, Scott Gladstein, Luay Almassalha, Greta Bauer, John Chandler, Lusik Cherkezyan, Di Zhang, Hariharan Subramanian, Igal Szleifer, Vadim Backman. A novel spectroscopic technology to image the native chromatin nanostructure in live cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A25.

78 Reversal of Hypermethylation As a Novel Strategy for NSAID Chemoprevention of Colon Carcinogenesis: Potential Role of Dnmt1

BACKGROUND AND AIM: Endoscopy is the first level procedure in the diagnosis of gastrointestinal (GI) masses. However gastro-duodenal or colonic lesions which mainly involve submucosa or subserosa as well as small bowels lesions may be difficult to diagnose. The present study aimed to evaluate the role of ultrasound (US)-guided percutaneous biopsy in diagnosing GI tract lesions. PATIENTS AND METHODS: 114 patients (63 male 51 female patients, age range 55-80 years, median age 68 years) underwent US-guided biopsy of GI lesions from 2000 to 2012. In 36 patients the lesion was in the small bowel, thus endoscopically inaccessible; in 50 patients previous histology of endoscopic biopsies was repeatedly negative; in 18 patients, stenosis did not allow bioptic sampling and in 10 patients age, severe cardiological disease and sepsis prevented endoscopy. We used multi-frequency convex probes with side adapters and 18G cutting needle for histological sampling. After biopsy, patients were monitored by measuring blood pressure and pulse rate during the following 3-4 hours to evaluate possible complications. Histology was compared with postsurgery histological evaluation in 73 cases. In the remaining, histology of US-guided biopsy was the only clue to diagnosis. RESULTS: Biopsy specimens were taken from stomach in 38 cases, small bowel in 36 cases and colon-sigma in 40 cases. Final diagnosis was malignant lesions in all but three cases. One case was intestinal endometriosis, the second was bowel duplication, whereas in the third case it was a false-negative result, the correct diagnosis being gastric adenocarcinoma. Sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of biopsy were respectively 99%, 100%, 100%, 66%, 99%.We observed no mortality and one complication: A case of melena due to bleeding from gastric gastrointestinal stromal tumor that was effectively controlled by positioning of metal clips. CONCLUSION: US-guided percutaneous biopsy of GI tract lesions may be an alternative, safe and effective procedure for diagnosis of GI tract lesions in those cases where conventional diagnostic approach is not feasible or successful.

134 Brahma-Related Gene 1 (BRG1) as a Novel Epigenetic Modulator of Gene Dysregulation in Early Colorectal Carcinogenesis: Implications for Chemoprevention

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

Su1871 Lactate Dehydrogenase-a Induction as an Early Metabolomic Marker of Colon Carcinogenesis: Potential Target for Chemoprevention

Our current study investigated the effect of PHLPP expression on the efficacy of chemotherapeutic drugs in colon cancer cells. Both PHLPP isoforms, PHLPP1 and PHLPP2, were either knocked down or overexpressed in SW480 and HT29 human colon cancer cells, and the rate of cell proliferation and apoptosis were measured upon drug treatment. Specifically, the cells were treated with different concentrations of oxaliplatin, PI3K inhibitor (LY294002), or rapamycin for 48 hours and cell proliferation was determined using MTS assays. Our results showed that knockdown of both PHLPP isoforms resulted in a decrease in the sensitivity to all drugs tested, whereas overexpression of PHLPP enhanced the anti-proliferation effect of these drugs. Moreover, we assessed the effect of the chemotherapy drugs in inducing cell death by monitoring the appearance of apoptotic markers including cleaved PARP and Caspase-3. Similarly, knockdown of both PHLPP isoforms rendered the cells resistant to apoptosis upon drug treatment. In summary, we have identified PHLPP as a critical factor in determining the drug sensitivity in the chemotherapeutic treatment of colon cancer. Adding yet another novel function of PHLPP and reasoning for the possible use of PHLPP as a therapeutic target in colon cancer.