Early Stages Of Carcinogenesis Research Articles

Abstract 5167: Expression levels of orexin receptor 1 in different stages of colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer death in Chile. Currently, treatment is surgical and adjuvance is based on chemotherapy mainly. One of the risk factors of CRC is obesity and metabolic syndrome. In search of factors that induce cell apoptosis, orexins were identified. Orexins are neuropeptides that regulate appetite, inhibit satiety and increase energy expenditure. The altered expression of orexin or its dysregulation has been associated with a wide range of human diseases such as narcolepsy, obesity, drug addiction and cancer. In rats and mice lacking orexin receptor, an increase in body mass index (BMI) was observed. Orexin Receptor 1 (OX1R) is overexpressed in CRC, but is not detected in normal colon tissue. Its activation by Orexin-A promotes apoptosis in cancer cell lines, and can be modulated by diet. Aim: To determine OX1R expression in the adenoma-carcinoma progression of CRC as possible therapeutic target, and its correlation with BMI. Material and Methods: Patients with neoplastic colorectal lesions undergoing surgical or endoscopic treatment between 2014 and 2015 were prospectively enrolled. Tissue samples of 29 patients were divided into 4 groups. Group A: control (n = 5), Group B: low-grade adenoma (n = 5), Group C: high-grade adenoma (n = 7), Group D: adenocarcinoma (n = 12). Formalin-fixed-paraffin-embedded samples were used to perform immunohistochemistry (IHC) of OX1R. Fresh tissues from Group D were used to assess protein and mRNA expression of OX1R by Western-blot and quantitative RT-PCR, respectively. Primary cultures were established from fresh tumor tissue of patients with adenocarcinoma. To compare OX1R expression levels we used the t-student test. Results: IHC expression of OX1R was detected both in the luminal membrane of colorectal epithelium and in the cytoplasm. We observed stained cells in 0-1% of the total area in Group A, B and C. In Group D, 7 samples showed staining 0-1%, 2 samples 1-10% and 3 samples 10-20%. High mRNA and protein expression was detected in stage III- IV adenocarcinoma samples compared to stage 0, I and II tumor samples. Normal adyacent tissue of CRC patients does not express OX1R. BMI is inversely associated with OX1R expression. Orexin A was able to induce apoptosis in primary cell cultures from advanced tumors in a dose dependent manner. Conclusions: OX1R is expressed scarcely in early stages of carcinogenesis. Its expression is induced significantly only in the most advanced adenocarcinomas, both at protein and mRNA levels, and is inversely associated with BMI. In primary cultures of patients with CRC, orexin A is able to induce apoptosis in a dose dependent manner. FONDECYT 1140012. Citation Format: Ana M. Wielandt, Cynthia Villarroel, Claudia Hurtado, Kento Inada, Hiroshi Kawachi, Daniela Simian, Maria T. Vial, Marcela Figueroa, Magdalena Castro, Udo Kronberg, Francisco Lopez-Kostner. Expression levels of orexin receptor 1 in different stages of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5167.

Non-neoplastic Fallopian Tube Epithelium Carrying Gene Mutations of a Novel SOX2 Repressor Region is Soil of High-grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) has a high mortality rate because the disease is asymptomatic in early stages and is resistant to treatments. A novel modality for diagnosis in the early stages of carcinogenesis is needed. In this issue of EBioMedicine, Hellner et al. present novel genetic aspects of small lesions of HGSOC (Hellner et al., 2016). They performed screening by LFR WGS technology for small HGSOC lesions from a patient who underwent treatment and found 750 genomic mutations. Gene ontology analysis revealed that the gene mutations are related to stem cell differentiation, and the authors finally found 6 novel non-coding mutations (BB1 - BB6) in proximity of sex determining region Y-box2 (SOX2), a key driver of stem cell differentiation (Takahashi and Yamanaka, 2006). Deep genetic analysis revealed that the BB5 region is commonly mutated in HGSOC tissues. A reporter assay and genome engineering using CRISPR/Cas9 system revealed that the BB5 region is a novel repressor of the SOX2 gene and that mutation in the BB5 region induces protein expression of SOX2. Mutations in the BB5 region were detected in non-neoplastic fallopian tube epithelium (FTE) of HGSOC cases and also in normal FTE of BRCA1 or BRCA2 mutation carriers who are at high risk for HGSOC. Surprisingly, immunohistochemical analysis of BB5 mutation carriers revealed that SOX2 protein is broadly expressed in even normal FTEs, indicating that non-neoplastic FTEs with BB5 mutation propagated clonally. SOX2 protein is expressed in earlier stages than TP53 protein expression indicating overexpression of SOX2 protein is earlier event than TP53 gene mutation. Interestingly, SOX2 protein expression decreased after the establishment of an ovarian cancer lesion. These results suggest that mutation in the SOX2 BB5 region is an early carcinogenesis driver mutation in HGSOCs and that detection of SOX2 protein is a promising biological marker for prediction of HGSOC.

The role of FOXA subfamily factors in embryonic development and carcinogenesis of the pancreas

The embryonic development and carcinogenesis are controlled by many transcription factors. The regulatory systems involved in embryogenesis of an organ are also involved in the tumor development in the same organ. FOX family proteins are transcription factors, which play a key role in these processes. The pioneering factors of the FOXA subfamily act at the very early stages of the embryonic development by interacting with condensed chromatin and thereby enabling the expression of the formerly silent important transcription factors. The role of these factors in tumor development is currently not fully elucidated, although recent studies indicate the important contribution of the FOXA subfamily proteins at the early stages of carcinogenesis. This review is restricted to the role of the FOXA factors in embryogenesis of the pancreas and their significance in the development of the pancreatic ductal adenocarcinoma.

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence.

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2′-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.

Telomere length shortening in gastric mucosa is a field effect associated with increased risk of gastric cancer.

Telomere shortening occurs in many organs and tissues and is accelerated by oxidative injury and rapid cell turnover. Short telomeres initiate chromosomal instability and may eventually contribute to tumorigenesis. To evaluate telomere length as potential biomarker for gastric cancer (GC) risk, we measured average telomere length using quantitative real-time PCR in GC tissues and in non-neoplastic mucosa from patients with GC and without GC. We obtained of 217 GC patients matched biopsies from the GC and adjacent tissues as well as gastric biopsies of 102 subjects without GC. Relative telomere length was measured in genomic DNA by real-time PCR. Relative telomere length decreased gradually in Helicobacter pylori (H. pylori) negative and positive gastric mucosa of GC free subjects compared with adjacent mucosa and cancer tissue from GC patients (4.03±0.3 vs. 2.82±0.19 vs. 0.82±0.07 vs. 0.29±0.09, P<0.0001). In non-neoplastic mucosa of GC patients, shorter telomeres were found significantly more often than in that of GC free subjects (age, sex, and H. pylori adjusted odds ratio=7.81, 95% confidence interval=4.71-12.9, P<0.0001). Telomere shortening in non-neoplastic mucosa was associated with chronic inflammation (P=0.0018) and intestinal metaplasia (P<0.0001). No significant associations were found between relative telomere length and clinicopathological features of GC and overall survival. Telomere shortening in gastric mucosa reflects a field effect in an early stage of carcinogenesis and is associated with an increased risk of GC. Telomere length in GC is not associated with clinicopathological features or prognosis.

Combined changes in Wnt signaling response and contact inhibition induce altered proliferation in radiation-treated intestinal crypts.

Curative intervention is possible if colorectal cancer is identified early, underscoring the need to detect the earliest stages of malignant transformation. A candidate biomarker is the expanded proliferative zone observed in crypts before adenoma formation, also found in irradiated crypts. However, the underlying driving mechanism for this is not known. Wnt signaling is a key regulator of proliferation, and elevated Wnt signaling is implicated in cancer. Nonetheless, how cells differentiate Wnt signals of varying strengths is not understood. We use computational modeling to compare alternative hypotheses about how Wnt signaling and contact inhibition affect proliferation. Direct comparison of simulations with published experimental data revealed that the model that best reproduces proliferation patterns in normal crypts stipulates that proliferative fate and cell cycle duration are set by the Wnt stimulus experienced at birth. The model also showed that the broadened proliferation zone induced by tumorigenic radiation can be attributed to cells responding to lower Wnt concentrations and dividing at smaller volumes. Application of the model to data from irradiated crypts after an extended recovery period permitted deductions about the extent of the initial insult. Application of computational modeling to experimental data revealed how mechanisms that control cell dynamics are altered at the earliest stages of carcinogenesis.

The number of regulatory Foxp3+ T-cells in different stages of malignant transformation of large intestinal polyps

PurposeThe aim of this study was to determine the relationship between the number of regulatory T-cells (Tregs) at various stages of malignant transformation of large intestinal polyps. Material/methodsThe study included tissue specimens from individuals subjected to complete colonoscopy with polypectomy and from patients who underwent surgical resection of colorectal tumors. This group included 27 individuals, among them 10 women (37%). Median age of the patients was 64 years (range 37–82 years). Surgical specimens included hyperplastic polyps (n=4), adenomatous polyps with low- (n=5) and high-grade dysplasia (n=8) and invasive colorectal cancers (n=10). Tregs were identified immunohistochemically. ResultsMean number of Foxp3+ T-cells per 10 high-power fields (HPFs) increased in line with malignant transformation, from 12.5 for hyperplastic polyps, 29.4 and 36.5 for adenomatous polyps with low- and high-grade dysplasia, respectively, to 56.3 for invasive colorectal cancers (p=0.00). An increase in the mean number of CD4+ T-cells was also observed, from 45.75, 57.8, 84.125, to 110.6 per 10 HPFs, respectively, however this change did not prove to be statistically significant (p=0.13). Mean Foxp3+/CD4+ T-cell ratio increased in line with malignant transformation (from 0.27, 0.3, 0.43, to 0.5), although a statistically significant change of this parameter was only observed in the case of invasive colorectal cancers (p=0.01). ConclusionsAn increase in the number of Tregs in the lymphocytic infiltrate of large intestinal polyps is interestingly already observed at early stages of carcinogenesis. Proportions of various T-cell subpopulations in the infiltrate vary considerably depending on the degree of dysplasia, especially in the case of invasive colorectal cancer.

MicroRNA alteration and putative target genes in high-grade prostatic intraepithelial neoplasia and prostate cancer: STAT3 and ZEB1 are upregulated during prostate carcinogenesis.

We aimed to identify alteration of cancer-related miRNAs in HGPIN and PCa, and to investigate the clinical implications of HGPIN as a precancerous lesion of PCa. Clinicopathologic analysis based on the status of HGPIN was performed in 388 patients who received radical prostatectomy between January 2005 and December 2008 in Severance Hospital. Among them, 10 paired HGPIN and PCa were prepared to perform miRNA microarray and quantitative real-time PCR. Fifty-two prostatectomy specimens were used to further validation of protein expression that was assessed by immunohistochemical staining (IHC) in matched non-neoplastic prostatic tissue (NPT), HGPIN, and PCa. Functional analysis was performed using a prostate normal cell line (RWPE-1) and two prostate cancer cell lines (LNCaP, PC-3) for comparison of expression of miR-155 and STAT3 mRNA before and after treatment of miR-155 mimetics/antagomir into each cell line. Patients with HGPIN had significantly less lymphovascular invasion, less lymph node metastasis, lower tumor volume, lower Gleason score, lower incidence of death, and longer overall survival compared to patients without HGPIN. MiR-155, miR-210, miR-153, and miR-200c were downregulated in HGPIN and PCa in common, compared to NPT. As putative target mRNAs, mRNA expression level of STAT3, ZEB1, and BACH1 was increased in PCa and HGPIN compared to NPT. mRNA expression level of ephrin-A3 was increased in PCa compared to NPT, and FGFRL1 was decreased in PCa compared to HGPIN and NPT. Protein expression assessed by IHC showed correlated results in STAT3, ZEB1, and ephrin-A3. Moreover, STAT3 and ZEB1 increased in a stepwise manner, from NPT to PCa. Treatment of miR-155 antagomir increased STAT3 mRNA expression in RWPE-1 cells, whereas treatment of miR-155 mimetics into PC-3 cells significantly decreased STAT3 expression. STAT3 and ZEB1 could be the key molecules altered at the early stages of carcinogenesis, especially in HGPIN. Prostate 76:937-947, 2016. © 2016 Wiley Periodicals, Inc.

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.

Abstract A14: Hedgehog inhibition delays cancer growth in C3(1)/Tag transgenic murine mammary carcinoma model

Abstract In 1941 Apperly hypothesized that differential sunlight exposure is the cause of the latitudinal gradient in breast cancer mortality, and subsequently the effect was postulated to be due to the tumor inhibitory properties of skin produced vitamin D3. We first tested this latitudinal hypotheses using the C3(1)/Tag transgenic murine mammary carcinoma model and found that ultraviolet radiation (UVR) significantly prolongs tumor-free and overall survival but that dietary D3 has no such effect. The mechanism of this anti-mammary carcinogenesis effect of UVR is unclear but there is evidence that D3 is a potent inhibitor of the hedgehog (HH) - signaling pathway. To test this mechanistic hypothesis we compared the effects of the small molecule Smo-acting HH pathway inhibitor (HHI) BMS-833923/XL139 with those of UVR on breast cancer development in C3(1)/Tag mice. We have found that oral administration of the pharmacologic HHI delays Tag mammary carcinogenesis with an efficacy similar to that of UVR treatment (350 mJ/cm2, 3x/week), thus, time to first palpable tumor was delayed by 9 days by UVR treatment (p=0.016) and by 14 days by HHI treatment (p=0.009), compared control mice. Overall survival was delayed by 9 days by UVR treatment (p=0.19) and by 15 days by HHI treatment (p=0.011). And, we have found that HHI, like UVR, acts at an early stage of carcinogenesis (i.e. reduction of histologic changes at age 12 weeks), and initial studies of UVR + HHI treatments suggest that HHI and UVR have overlapping effects. Taken together our results are consistent with (albeit by no means proof of) our hypothesis that inhibition of mammary carcinogenesis by UVR-produced vitamin D3 underlies the latitudinal gradient and is mediated via HH inhibition. Moreover, our results suggest more broadly that HHI treatment before tumors develop may have more of an anti-cancer effect than does HHI treatment after tumors already have become clinically apparent. Note: This abstract was not presented at the conference. Citation Format: Anastasia M. Makarova, Subheksha KC, John A. Dolorito, Yefim I. Khaimskiy, Ervin H. Epstein, Jr. Hedgehog inhibition delays cancer growth in C3(1)/Tag transgenic murine mammary carcinoma model. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A14.

The expression of IGF-1R in Helicobacter pylori-infected intestinal metaplasia and gastric cancer.

Overexpression of IGF-1R has been demonstrated in gastrointestinal cancers, and its expression is reported as the result of the loss of tumor suppressors. IL-16 is involved in the pathophysiological process of chronic inflammatory diseases. The aim of this study is to determine the changes in the expression of IGF-1R in intestinal metaplasia (IM) and gastric cancer (GC) as well as the effect of Helicobacter pylori (H. pylori) and IL-16 on cell proliferation and IGF-1R expression in gastric cells. AGS cells were incubated with combinations of IL-16 and H. pylori. Gastric cell proliferation was studied by BrdU uptake. In H. pylori infected mucosa, IGF-1R was significantly higher in IM than chronic gastritis (CG), and also higher in GC than CG and IM. H. pylori significantly decreased BrdU uptake. IL-16 increased BrdU uptake and IGF-1R on AGS cells which had been decreased by H. pylori. Co-incubation with IL-16 increased the expression of IGF-1R mRNA in H. pylori infected cells. We conclude that the expression of IGF-1R in H. pylori infected gastric mucosa may indicate an early stage of carcinogenesis. The IL-16 secretion by H. pylori can be a trigger for the expression of IGF-1R, and it may also be a factor for gastric carcinogenesis.

Environmental Toxicant Exposure and Cancer: The Role of Epigenetic Changes and Protection by Phytochemicals.

Exposure to environmental toxicants is a well-documented predisposing factor for cancer. Although genetic alterations have long been known to occur through exposure to some environmental carcinogens, there is another layer of genome regulatory system named epigenetic system. Epigenetics is defined as any reversible and heritable change in cellular patterns of gene expression that does not alter DNA sequence. This layer of gene control plays a key role in early stages of carcinogenesis by reprogramming cells to what is known as cancer stem cells, a process with great similarities to somatic cell reprogramming into "induced pluripotent stem cell". Environmental toxicants could directly promote carcinogenesis through disturbing promoter CpG island hypermethylation, and silencing of tumor suppressor genes, hypomethylation of transposable elements and genomic instability induced by environmental toxicants. Environmental toxicants could also indirectly affect epigenetic programming of nucleus through inducing inflammatory signaling pathways that converge on NF-κB or STAT3 activation. Considering the reversibility of epigenetic alterations and their pivotal role in early carcinogenesis, reversion of these alterations could be a promising approach for chemoprevention. Selected phytochemicals have shown desirable effects through regulation of the most important epigenetic mechanisms including DNA methylation, histone modifications and microRNA expression, as well as modulation of SIRT-1 and STAT-3 signaling pathways. The present review aims to outline the epigenetic mechanisms underlying carcinogenic effects of environmental toxicants, and the protective effects of phytochemicals in reversing epigenetic aberrations in the regulatory pathways steering normal cell homeostasis.

Polychlorinated Biphenyls and Cancer: Are Telomeres to Blame?

The accumulating epidemiological evidence of elevated cancer risk and mortality in individuals exposed to polychlorinated biphenyls (PCBs) led to their recent classification as human carcinogen by the International Agency for Research on Cancer (IARC) (International Agency for Research on Cancer (IARC), 2015). However, the mechanisms by which PCBs are linked to cancer are still unclear. In this issue, Scinicariello and Buser (Scinicariello, 2015) conducted a study of the association between leukocyte telomere length (LTL) and PCB blood levels in a nationally representative sample of the civilian US adult population using data from the National Health and Nutrition Examination Survey (NHANES). The authors showed that higher PCB blood levels were associated with longer LTL, and hypothesized that there could be a link to PCB-related carcinogenesis. A separate study reported similar relationships between LTL and PCB blood levels in a subset of NHANES participants (Mitro et al., 2015). However, a small study of healthy Koreans (Shin et al., 2010) showed that this relationship was only present at low PCB levels. In contrast, short LTL has been associated with a number of environmental or occupational exposures including particulate matter, black carbon, benzene, toluene, polycyclic aromatic hydrocarbons, N-nitrosamines, pesticides, and lead (reviewed in Zhang et al., 2013), all of which may contribute to PCB blood levels. Telomere shortening in response to chemical exposures may be explained, at least in part, by the induction of an oxidative stress DNA damage response (Zhang et al., 2013). Telomeres, the long (TTAGGG)n nucleotide repeats and an associated protein complex at chromosome ends, are essential for maintaining chromosomal stability. They shorten with each cell division and therefore are markers for cellular replicative capacity, cellular senescence, and aging. Telomere shortening and chromosomal instability has been described at early stages of carcinogenesis, suggesting a role of telomere dysfunction in cancer initiation (Ferguson et al., 2015). Cancer epidemiology studies have shown relationships with short LTL, but differences by cancer site and study design were noted. In a meta-analysis of 27 reports (Wentzensen et al., 2011), short LTL was associated with cancer risk, primarily in case–control studies (OR = 2.9 in case–control studies vs. 1.2 in prospective studies). This suggests that short LTL is a disease marker rather than a risk factor. Recently, a large study linked long LTL to melanoma risk (Iles et al., 2014). The spectrum of cancers associated with long LTL is not clear yet, but suggested to include lung and Non-Hodgkin's lymphoma (NHL). It is of interest that cutaneous melanoma and possibly NHL are the main cancers associated with excess risk in PCBs exposed individuals (International Agency for Research on Cancer (IARC), 2015), suggesting a possible contribution of LTL in PCB-related carcinogenesis. The large sample size, the national representation, and the wealth of collected information NHANES offers are among the strengths of this study. On the other hand, the cross-sectional design restricted its ability to establish a causal inference between LTL, PCB exposure, and cancer risk. The one-time measurement of PCB blood level and LTL may not reflect the full spectrum of the association. Information on the duration and dose of exposure to PCBs would have been useful (reviewed in Zhang et al., 2013) Short-term exposure to ambient particulate matter was associated with long LTL, while longer exposures were associated with short LTL. Exposure dose in relation to LTL was noted in other environmental exposures. For example, exposure to low dose of arsenic resulted in telomerase overexpression and telomere elongation in cord blood cells, while exposure at large doses suppressed telomerase, shortened telomeres, and induced apoptosis. A recent study (Andreotti et al., 2015) evaluating the association between LTL and pesticide exposure suggested a differential effect on LTL in long-term versus recent exposures for certain pesticides. While LTL may present a good surrogate for telomere length in other tissues, it is not clear how good of a surrogate it is for tissues affected by chemical exposures. Measuring telomere length in organs directly affected by PCB exposure, and comparing this to LTL may explain the role telomeres may play in PCB-related carcinogenesis. Also, LTL measurement obtained by quantitative polymerase chain reaction (qPCR) assay may be affected by the cell composition of the sample since telomere length differs by peripheral blood cell subtype. Using a leukocyte cell-type specific TL measurement assay such as flow FISH may provide better insights into such association. Important considerations in epidemiological studies of telomere length are discussed in details elsewhere (Bodelon et al., 2014). In conclusion, the mechanism behind PCB-related carcinogenesis is still unknown. The similarity between cancer sites associated with long LTL, and cancers linked with PCBs provides an interesting hypothesis to test. A large prospective study with serial LTL and PCBs level measurements is required to better understand this relationship.

Genetic-deletion of Cyclooxygenase-2 Downstream Prostacyclin Synthase Suppresses Inflammatory Reactions but Facilitates Carcinogenesis, unlike Deletion of Microsomal Prostaglandin E Synthase-1.

Prostacyclin synthase (PGIS) and microsomal prostaglandin E synthase-1 (mPGES-1) are prostaglandin (PG) terminal synthases that function downstream of inducible cyclooxygenase (COX)-2 in the PGI2 and PGE2 biosynthetic pathways, respectively. mPGES-1 has been shown to be involved in various COX-2-related diseases such as inflammatory diseases and cancers, but it is not yet known how PGIS is involved in these COX-2-related diseases. Here, to clarify the pathophysiological role of PGIS, we investigated the phenotypes of PGIS and mPGES-1 individual knockout (KO) or double KO (DKO) mice. The results indicate that a thioglycollate-induced exudation of leukocytes into the peritoneal cavity was suppressed by the genetic-deletion of PGIS. In the PGIS KO mice, lipopolysaccharide-primed pain nociception (as assessed by the acetic acid-induced writhing reaction) was also reduced. Both of these reactions were suppressed more effectively in the PGIS/mPGES-1 DKO mice than in the PGIS KO mice. On the other hand, unlike mPGES-1 deficiency (which suppressed azoxymethane-induced colon carcinogenesis), PGIS deficiency up-regulated both aberrant crypt foci formation at the early stage of carcinogenesis and polyp formation at the late stage. These results indicate that PGIS and mPGES-1 cooperatively exacerbate inflammatory reactions but have opposing effects on carcinogenesis, and that PGIS-derived PGI2 has anti-carcinogenic effects.

Abstract 1048: Chromatin remodelers are frequently disrupted by epigenetic and genetic alterations at an early stage of carcinogenesis

Abstract Chromatin remodeling factors (chromatin remodelers), such as SWI/SNF and ISWI complexes, regulate gene transcription by modulating chromatin accessibility. Genes encoding subunits of chromatin remodelers are frequently disrupted by loss of function mutations in various types of cancers. In contrast, inactivation of chromatin remodelers by epigenetic alterations, such as aberrant DNA methylation, is unknown. In this study, we aimed to clarify 1) whether or not chromatin remodelers are inactivated by aberrant DNA methylation, and 2) the timing of this occurrence. DNA methylation statuses of 16 genes encoding chromatin remodelers with promoter CpG islands were analyzed in non-cancerous tissues of male gastric cancer patients using an Infinium HumanMethylation450 BeadChip array. Among the 16 genes, two genes, ACTL6B and SMARCA1, were frequently methylated in non-cancerous gastric tissues. Among these genes, SMARCA1 was expressed at high levels in normal gastric tissues, but ACTL6B was not. In four of 50 primary gastric cancers, SMARCA1 was aberrantly methylated. It was re-expressed after treatment with a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC), in gastric cancer cell lines, AGS and GC2. Somatic mutations of 18 genes encoding chromatin remodelers were analyzed using a next-generation personal sequencer in the 50 primary gastric cancers. Nine genes, ARID1A, ARID1B, ARID2, SMARCA1, SMARCA2, SMARCA4, SMARCB1, PBRM1, and PHF10, were mutated in one or more of the 50 gastric cancers. The mutant allele frequency was close to the cancer cell content in cancer samples with a specific mutation of a chromatin remodeler. Depletion of a chromatin remodeler, SMARCA1 or SMARCA2, by shRNAs in cancer cell lines promoted their growth. These results showed that epigenetic and genetic alterations of chromatin remodelers are induced at an early stage of carcinogenesis (Takeshima et al., Cancer Letters, in press). Citation Format: Hideyuki Takeshima, Tohru Niwa, Takamasa Takahashi, Takayuki Ando, Hirokazu Taniguchi, Hitoshi Katai, Toshiro Sugiyama, Toshikazu Ushijima. Chromatin remodelers are frequently disrupted by epigenetic and genetic alterations at an early stage of carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1048. doi:10.1158/1538-7445.AM2015-1048

Abstract 4033: Suppressive role of normal epithelium in pre-malignant to malignant progression of Barrett's esophagus

Abstract Cell-cell communication plays a central role in cellular function under normal and diseased conditions. In cancer, intercellular interactions are highly complex and remain poorly understood, particularly with respect to the early events leading to the transformation from benign to malignant phenotypes. In early stages of carcinogenesis, normal and transformed cells often coexist in close proximity, suggesting that interactions between them influence progression. Comprehensive understanding of these events is critical in identifying new therapeutic targets and devising novel treatment strategies. We studied the functional relevance of intercellular interactions between normal and pre-malignant epithelial cells using an in vitro model of Barrett's esophagus (BE) progression from pre-malignant epithelium to esophageal adenocarcinoma. BE provides an ideal disease model to explore the interaction between cells at different stages of neoplastic transformation because it can be prospectively followed. Thus, studies performed in in vitro and animal models can be readily extended to the clinic. We investigated the effects of heterotypic interactions between normal and dysplastic (pre-malignant) esophageal epithelial cells at the single-cell and bulk-cell level. We found that the dysplastic cells were affected to a larger extent than normal cells in terms of gene expression levels and cellular function. Analysis of the RNA-seq data revealed a total of 809 genes differentially expressed in dysplastic cells as compared with 234 genes differentially expressed in normal cells. Functional enrichment analysis identified the following 5 top functional categories in dysplastic cells affected (downregulated) by interactions with normal epithelial cells: cellular movement, tissue morphology, cancer, lipid metabolism and molecular transport. Functional assays revealed a significant repression of the proliferation rates (up to 6-fold) of both cell types, but only dysplastic cells showed significant motility changes (speed, displacement and directionality) as a result of interactions between the two cell types. The decrease in the proliferation rate of dysplastic cells indicates a strong suppressor role of normal epithelial cells mediated by repression of TGFβ and EGF signaling in pre-malignant progression in BE. At the single-cell level, we present both metabolic and expression-level profiles of a panel of selected genes in individual normal and dysplastic cells, and compare them with bulk-cell measurements. These findings indicate that attenuation of the TGFβ and EGF signaling pathways in the presence of normal epithelial cells may play an important role in suppressing the growth of pre-malignant epithelial cells. Hence, both TGFβ and EGF may serve as biomarkers for progression from pre-malignant to malignant phenotype in epithelial cells. Citation Format: Laimonas Kelbauskas, Jia Zeng, Aida Rezaie, Kristen Lee, Benjamin Ueberroth, Weimin Gao, Dmitry Derkach, Colleen Ziegler, Thai Tran, Kimberly J. Bussey, Dean Smith, Roger H. Johnson, Deirdre R. Meldrum. Suppressive role of normal epithelium in pre-malignant to malignant progression of Barrett's esophagus. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4033. doi:10.1158/1538-7445.AM2015-4033

Abstract 4833: Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration

Abstract In human cervical carcinogenesis, the gain of the long arm of chromosome 3 is the decisive genetic event that determines progression of dysplasia to invasive disease. Other recurrent genomic imbalances, such as gains on 1q, 5p, 8q, and 20q, and losses on 2p, 3p, and 11q are also observed. To better understand the patterns of clonal evolution during disease progression we developed a multiplex FISH assay with three multicolor probe panels for sequential hybridizations of cervical intraepithelial neoplasia (CIN3) and invasive cervical carcinoma (ICC), including synchronous CIN3/ICC lesions. Our FISH assay targets oncogenes COX2 (1q), TERC (3q), TERT (5p), MYC (8q), ZNF217 (20q), and tumor suppressor genes ING5 (2p), FHIT (3p), CHEK1 (11q), and centromere 7 as a ploidy control. Our results confirmed the dominant role of the gain of 3q: nineteen out of twenty lesions showed a major clonal population with TERC amplification. Nine out of the 20 lesions showed a concomitant loss of FHIT (3p) suggesting an isochromosome 3q formation, which makes the loss of FHIT the second most frequent clonal event in this group of samples. Other imbalances were present, but, as expected, less common. The copy number increases of 3q can occur either in diploid cells or after tetraploidization of the genome. Interestingly, in our samples only three CIN3 lesions showed major clones with signal patterns consistent with near-tetraploidy, while all other lesions, including all ICC lesions and the two synchronous CIN3/ICC lesions showed major signal patterns consistent with near-diploid cell populations. Although tetraploidization can occur already in premalignant lesions, and there is independent evidence that such chromosome doublings increase the risk of chromosome segregation errors, and therefore promote the subsequent acquisition of genomic imbalances, our data indicate that a majority of cervical precancerous and cancerous lesions acquire aberrations in a diploid genome. Independent of whether the 3q gain and other aberrations are acquired in a diploid or tetraploid background, it selects cells with a proliferative advantage, which is supported by our observations that (i) cells with identical patterns of 3q gain are adjacent to each other, hence indicating a clonal proliferation, and (ii) that the only commonality between the major clones in the synchronous CIN3 and ICC lesions was a TERC gain. We further examined overall cell-to-cell heterogeneity in CIN3 versus ICC samples. While overall levels of clonal heterogeneity were indistinguishable between CIN3 and ICC, phylogenetic models of single tumors suggested greater average numbers of mutations per cell in ICC versus CIN3. These findings improve our understanding of clonal development during cervical carcinogenesis and with that our understanding of genome dynamics at early stages of carcinogenesis. Citation Format: Leanora S. Hernandez, Amanda Bradley, Timo Gaiser, Sonia Andersson, E. Michael Gertz, Salim A. Chowdhury, Russel Schwartz, Alejandro Schäffer, Kerstin Heselmeyer-Haddad, Thomas Ried. Single-cell genetic analysis reveals insights into clonal development of cervical cancer and confirms TERC as an early and dominant aberration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2015-4833

Sustained NRF2 activation in hereditary leiomyomatosis and renal cell cancer (HLRCC) and in hereditary tyrosinemia type 1 (HT1).

The nuclear erythroid 2-like 2 transcription factor (NRF2), is a major regulator of cellular redox balance. Although NRF2 activation is generally regarded as beneficial to human health, recent studies have identified that sustained NRF2 activation is over-represented in many cancers. This raises the question regarding the role of NRF2 activation in the development and progression of those cancers. This review focuses on the mechanisms and the effects of NRF2 activation in two hereditary cancer predisposition syndromes: hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary tyrosinemia type 1 (HT1). Because the cancer initiating mutations in these hereditary syndromes are well defined, they offer a unique opportunity to explore the roles of NRF2 activation in the early stages of carcinogenesis. Over the years, a variety of approaches have been utilized to study the biology of HLRCC and HT1. In HLRCC, in vitro studies have demonstrated the importance of NRF2 activation in sustaining cancer cell proliferation. In the mouse model of HT1 however, NRF2 activation seems to protect cells from malignant transformation. In both HT1 and HLRCC, NRF2 activation promotes the clearance of electrophilic metabolites, enabling cells to survive cancer-initiating mutations. Biological insights gained from the hereditary syndromes' studies may shed light on to the roles of NRF2 activation in sporadic tumours.

The potential role of microRNA-31 expression in early colorectal cancer.

The expression of microRNA-31 (miR-31) has been implicated in the progression of some human malignancies including colorectal cancer. However, the clinical significance of the expression of miR-31 in submucosally invasive (T1) colorectal cancer remains unclear. The aim of the present study was to delineate the relationship between clinicopathological features and the oncogenic modulator miR-31 in submucosally invasive colorectal cancer. We investigated the expression of miR-31 in 50 submucosally invasive colorectal cancer specimens, along with the corresponding non-tumoral mucosa specimens, using a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The relationships between miR-31 expression levels and clinicopathological characteristics were assessed. The miR-31 host gene locus was investigated using fluorescence in situ hybridization. qRT-PCR revealed that the expression of miR-31 was higher in colorectal cancer tissue than in non-tumoral tissue (P = 0.0002). The up-regulated expression of miR-31 may play an oncogenic role in the early stage of carcinogenesis in colorectal cancers.

Areca nut exposure increases secretion of tumor-promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes.

Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)‐exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT‐hNOF) was used. We found that the levels of GRO‐α, IL‐6 and IL‐8 increased in AN‐exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN‐exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8‐oxoG FITC‐conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN‐exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine‐triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.