Early Tumorigenesis Research Articles

Effect of ARID1A/BAF250a expression on carcinogenesis and clinicopathological factors in pure-type clear cell adenocarcinoma of the ovary.

Frequent mutation of the ARID1A gene has been recently identified in ovarian clear-cell adenocarcinoma (CCA); however, the clinical significance of BAF250a expression encoded by the ARID1A gene remains to be determined. The aim of the present study was to assess whether BAF250a expression had an impact on the clinical features of CCA. A total of 97 cases of CCA treated at a single institution were enrolled in the present study. The tissue samples were evaluated by immunohistochemical staining. BAF250a-deficient expression was observed in 30% (29/97) of all CCA cases. Of this, 19% of non-atypical endometriosis, 26% of atypical endometriosis, 39% of endometriosis-related CCA, 5% of benign clear-cell adenofibroma (CCAF), 5% of borderline CCAF and 10% of CCAF-related CCA. BAF250a-deficient expression was significantly more frequent in endometriosis-related CCA compared with that in CCAF-related CCA (P=0.02). No significant difference was observed in the response rate of primary chemotherapy according to BAF250a expression status (P=0.48). Additionally, BAF250a expression status was not significantly correlated with progression-free and overall survival in patients with CCA. Although loss of BAF250a expression was associated with early tumorigenesis in endometriosis-related CCA, this alteration was not significantly correlated with chemosensitivity and prognoses of CCA. Further biomarker analyses, including BAF250a expression, are required to improve the prognoses of CCA.

Analysis of different components in the peritumoral tissue microenvironment of colorectal cancer: A potential prospect in tumorigenesis.

The present study aimed to observe the varying expression of biomarkers in the microenvironment adjacent to colorectal cancer lesions to provide additional insight into the functions of microenvironment components in carcinogenesis and present a novel or improved indicator for early diagnosis of cancer. A total of 144 human samples from three different locations in 48 patients were collected, these locations were 10, 5 and 2 cm from the colorectal cancer lesion, respectively. The biomarkers analyzed included E-cadherin, cytokeratin 18 (CK18), hyaluronidase-1 (Hyal-1), collagen type I (Col-I), Crumbs3 (CRB3), vimentin, proteinase activated receptor 3 (PAR-3), α-smooth muscle actin (α-SMA), cyclin D1 (CD1) and cluster of differentiation (CD)133. In addition, crypt architecture was observed. Related functional analysis of proteins was performed using hierarchical index cluster analysis. More severe destroyed crypt architecture closer to the cancer lesions was observed compared with the 10 cm sites, with certain crypts degraded entirely. Expression levels of E-cadherin, CK18, CRB3 and PAR-3 were lower in 2 cm sites compared with the 10 cm sites (all P<0.001), while the expression levels of the other biomarkers in the 2 cm sites were increased compared with 10 cm sites (all P<0.0001). Notably, the expression of CK18 in 2 cm sites was higher than in the 5 cm site (P<0.0001), which was different from the expression of E-cadherin, CRB3 and PAR-3. The expression levels of Hyal-1 and Col-I at the 2 cm sites were lower than that of the 5 cm sites (P>0.05 and P=0.0001, respectively), while the expression of vimentin, α-SMA, CD1 and CD133 were not. Hyal-1 and Col-I may be independently important in cancer initiation in the tumor microenvironment. The results of the present study suggest that the biomarkers in the tissue microenvironment are associated with early tumorigenesis and may contribute to the development of carcinomas. These observations may be useful for early diagnosis of colorectal cancer.

Abstract 4291: Vitamin B6 biomarkers and colorectal cancer: Modifications by time

Abstract Background: Pyridoxal 5’-phospate (PLP) is the most commonly used marker of circulating vitamin B6 status. In prospective case-control studies, individuals with higher plasma concentrations of PLP had a 30-50% decreased risk of colorectal cancer (CRC). This may partly be explained by decreased PLP due to inflammatory activity in persons at an increased risk of CRC. We aimed to investigate different markers of vitamin B6 in relation to CRC risk in a population with a very long follow-up time between screening and diagnosis. Methods: This was a prospective case-control study of 613 incident CRC cases and 1190 matched controls nested within the population-based Northern Sweden Health and Disease Study. PLP, pyridoxal (PL), pyridoxic acid (PA), 3-hydoxykynurenine (HK) and xanthurenic acid (XA) were measured in plasma by LC-MS/MS. Besides PLP, we investigated the recently introduced metabolite ratios PAr (PA/(PLP+PL)) and HK:XA as markers of vitamin B6 status in relation to CRC risk. Results: PAr, a marker of vitamin B6 catabolism during inflammation, was linearly associated with CRC risk, with a 50% higher risk in the highest vs. lowest quartile. In the group with a follow up time between screening and diagnosis over 10.5 years no association was observed, while in the groups with a follow up time between 5.8-10.5 years and below 5.8 years risk was approximately twofold in most quartiles vs the lowest quartile. A 50% risk decrease was observed in the in the third vs the lowest quartile of plasma PLP and a similar risk increase was observed in the highest vs the lowest quartile of the functional vitamin B6 marker HK:XA. Conclusions: PAr may influence later stages of tumor development, but possibly not early tumorigenesis. Poor functional vitamin B6 status and inflammatory activity might influence both the risk of CRC and plasma concentrations of PLP. Our results underscore the need to use several biomarkers for vitamin B6 status together with the already well established plasma PLP. This may be generalizable not only to CRC but also other diseases with an inflammatory aspect. Citation Format: Björn Gylling, Robin Myte, Jörn Schneede, Göran Hallmans, Jenny Häggström, Ingegerd Johansson, Arve Ulvik, Øivind Midttun, Per Magne Ueland, Bethany van Guelpen, Richard Palmqvist. Vitamin B6 biomarkers and colorectal cancer: Modifications by time. [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 4291.

Abstract 4175: ERG oncogenic activation leads to the endoplasmic reticulum stress and cell survival mechanisms

Abstract Introduction: High levels of the ERG expression due to gene fusions (predominantly TMPRSS2-ERG) is frequently detected in prostate cancer (CaP) in Western countries. Better understanding of the roles of the ERG oncogenic functions in CaP initiation and/or progression will enhance its utility as the therapeutic target. Transgenic mice generated to understand role of ERG in the CaP development have shown focal proliferative and dysplastic PIN lesions and adenocarcinoma in older mice. The present study was aimed towards understanding the molecular mechanism underlying the role of ERG in early stages of oncogenic transformation of prostate epithelium. Methods: Morphological analyses of mouse prostate glands were performed by H&amp;E staining and by electron microscopy. Luminal cell surface markers were evaluated by FACS analysis. Luminal cells were assessed for their potential to proliferate and form spheres by prostate sphere formation assay. Effects of ERG on ER stress and UPR marker proteins were analyzed in transgenic mice prostates and ERG expressing cell culture models. Representative ER stress pathway in ERG-positive human prostate cancer was analyzed by CPDR-Affymetrix GeneChip data from well and poorly differentiated tumors. Results: Histological examination of ERG-Tg mouse prostates revealed increased luminal cell death due to apoptosis. Subsequently, TEM analysis revealed significant morphological differences such as increased numbers of lysosomes, autophagosomes, concentric membrane bodies with ribosomes, and lipid droplets in the cytoplasm of transgenic secretory luminal epithelial cells. Epithelial cells of ERG-Tg mouse prostates showed increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to induced cell death. ERG-Tg mouse prostate tissues and LNCaP-ERG transfectants showed increased expression of ER stress sensors and UPR proteins. Further, in human prostate tumors, a strong correlation was also observed between expression of the ERG and P4HB/PDI, an ER stress response protein. Conclusions: A critical of function of ERG in early prostate tumorigenesis may involve ER stress resulting into the activation of UPR, autophagy and cell survival through clonal selection. These observation also define potential new therapeutic targets in CaP-ERG network. Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and USU-CPDR funds. Citation Format: Taduru L. Sreenath, Shiela Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Parameet Kumar, Denise Young, Rishita Gupta, Shilpa Katta, Ahmed Mohamed, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Charles J. Bieberich, Peter Nelson, David G. McLeod, Valeri Vasioukhin, Shiv Srivastava. ERG oncogenic activation leads to the endoplasmic reticulum stress and cell survival mechanisms. [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 4175.

Abstract 1028: A mouse model of allelic loss of the core autophagy gene BECN1 in ovarian cancer

Abstract Autophagy, a process of protein and organelle recycling, can both suppress and promote tumors. Gliomas, KRAS mutant, and BRAF driven tumor models require functional autophagy for maximal tumorigenicity and/or tumor progression. Inhibition of autophagy is able to eliminate many human cancer cell lines. However, allelic loss of the autophagy gene BECN1 is present in the majority of ovarian cancers, and BECN1 loss incurs early tumorigenesis in mouse models free of other oncogenic stimuli. Recent sequencing efforts and analyses, depending on the study, have both supported and refuted the relevance of BECN1 allelic loss in ovarian and breast cancers. To directly address the role of BECN1 allelic loss in ovarian cancer, we crossed BECN1 heterozygous knockout dams with MISIIR-TAg sires. In this model, the MISIIR promoter induces expression of large T antigen specifically in the ovarian and fallopian epithelium, inhibiting p53 activity and driving ovarian cancers in a manner befitting its human analogue. Here we present our pre-publication findings relating to the oncogenesis and progression of this murine BECN1 allelic loss model of ovarian cancer. We compare the in vivo murine results to those of in vitro human cell line models of beclin depletion, which exhibit higher basal dsDNA break repair signaling, mitochondrial stress, and reactive oxygen species generation. Citation Format: Joe R. Delaney, Chandni Patel, Dwayne G. Stupack. A mouse model of allelic loss of the core autophagy gene BECN1 in ovarian 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 1028.

Clinicopathological significance of p15 promoter hypermethylation in multiple myeloma: a meta-analysis.

Published studies reported that loss of function of the p15INK4B gene is caused by hypermethylation; however, whether or not the inactivation is associated with the incidence and clinical significance of multiple myeloma (MM) remains unclear. In this study, we performed a meta-analysis to quantitatively determine the effects of p15 hypermethylation on the incidence of MM. The related research articles in English and Chinese languages were evaluated. The data were extracted and assessed independently. The pooled data were analyzed and odds ratios were calculated and summarized. Sixteen eligible studies were selected for final analysis. We demonstrated that p15 hypermethylation is significantly higher in MM than that in normal bone marrow, as well as monoclonal gammopathy of undetermined significance. However, aberrant p15 hypermethylation was not significantly higher in advanced MM than that in early-stage MM. The results of this study reveal that p15 hypermethylation is correlated with an increased risk in the progression of monoclonal gammopathy of undetermined significance to MM. p15 hypermethylation, which induces the loss of function of the p15 gene, plays a critical role in the early tumorigenesis of MM and serves as a reputable diagnostic marker and potential drug target.

Interleukin-37 expression and its potential role in oral leukoplakia and oral squamous cell carcinoma.

Interleukin 37 (IL-37) has been reported to play a significant role in innate immune response and to be involved in several kinds of cancers. However, the investigation of association between IL-37 and oral mucosa carcinogenesis hasn't been clearly established. The aim of the study was to assess IL-37 expression and explore its role in oral mucosa carcinogenesis. The expression of IL-37 increased from normal control (NC) to Oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC). Moreover, statistically highly significant difference was present between scores of OLK with and without mild/moderate dysplasia (P < 0.001). In addition, IL-37 expression was lower in OSCC with lymph node metastasis than those without metastasis (P < 0.01). What’s more, overexpression of IL-37 in RAW264.7 cells remarkably reduced the pseudopodia, vacuolization and the expression of IL-6, TNF-α, and IL-1β. Finally, we found IL-37 and its receptor IL-18Rα but not its binding partner IL-18BP have similar tissue location and expression trend in different stages of oral mucosa carcinogenesis. Overall, IL-37 can be used as a biomarker for early oral tumorigenesis and for malignant transformation risk assessment of premalignant lesions.

An Approach to Breast Cancer Immunotherapy: The Apoptotic Activity of Recombinant Anti-Interleukin-6 Monoclonal Antibodies in Intact Tumour Microenvironment of Breast Carcinoma.

Current work is one of our comprehensive preclinical studies, a new approach to breast cancer (BC) immunotherapy through induction of tumour cell apoptosis. Tumour growth is not just a result of uncontrolled cell proliferation but also of reduced apoptosis. High levels of interleukin-6 (IL-6) are associated with metastatic BC and correlated with poor survival as it promotes growth of tumour-initiating cells during early tumorigenesis protecting these cells from apoptosis. Therefore, this study aims at investigating the potential of anti-IL-6 monoclonal antibodies to suppress IL-6 proliferative/anti-apoptotic activities in intact tumour microenvironment of BC. Fresh sterile tumour and normal breast tissue specimens were taken from 50 female Egyptian patients with BC undergoing radical mastectomy. A unique tissue culture system designed to provide cells of each intact tumour/normal tissue sample with its proper microenvironment either supplemented or not with anti-IL-6 monoclonal antibodies. To evaluate the apoptotic activity of anti-IL-6 as a novel candidate for BC treatment strategy, we compared its effects with those obtained using tumour necrosis-related apoptosis-inducing ligand TRAIL as an established apoptotic agent. Our results revealed that levels of either anti-IL-6- or TRAIL-induced apoptosis in the tumour or normal tissue cultures were significantly higher than those in their corresponding untreated ones (P<0.001). No statistically significant differences have been found between apoptosis levels induced by anti-IL-6 monoclonal antibodies and those induced by TRAIL. Recombinant anti-IL-6 monoclonal antibodies could represent a novel effective element of immunotherapeutic treatment strategy for BC. The selectivity and anti-apoptotic potential of anti-IL-6 is highly hopeful in IL-6- abundant BC tumour microenvironment.

Role of N-acetylgalactosaminyltransferase 6 in early tumorigenesis and formation of metastasis.

Glycosylation is one of the most important posttranslational modifications of proteins and lipids that contributes to the structural diversity of cellular molecules. Enzymes of the glycosyltransferase class are responsible for altering glycosylation patterns by adding carbohydrate chains to the respective acceptor molecules. It is well known that glycosylation is commonly altered in cancerous tissue. Therefore, the present study aimed to determine the incidence of N‑acetylgalactosaminyltransferase 6 (GALNT6), a prominent member of the glycosyltransferase class, in breast cancer tissue of different developmental stages by immunohistochemistry. Although no correlation was identified between tumour characteristics and GALNT6 staining intensity, to the best of our knowledge, this is the first study to demonstrate that tissue from carcinoma in situ‑tumours and metastases were more heavily stained than late‑stage breast cancers. This may indicate an important role of glycosylation aberration in escaping the immune system at early phases of tumour development. The present study also hypothesised that nascent or early metastasizing tumours are normally recognized by the immune system of the patient, but glycosylation pattern changes may facilitate tumor escape from immune recognition. In follow‑up studies, our group will aim to confirm and consolidate these results in a larger patient cohort that may give greater insight into breast cancer characterization as well as tumour treatment.

Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.

Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation.

To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1) neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs) and Schwann cells (SCs) prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3(fl/fl);Nf1(fl/fl);DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

Abstract A50: The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous disease of significant mortality and morbidity and with limited treatment options. This highlights the urgent need for a better understanding of the underlying biology of HNSCC. Recent genomic analysis of HNSCC tumors has identified four distinct molecular classes, of which the mesenchymal subtype is particularly aggressive in nature and associated with poor survival. We have discovered that Ets1, a prototypic oncogenic transcription factor, is selectively enriched in the mesenchymal HNSCC tumors along with other key drivers of Epithelial to Mesenchymal transition (EMT). Further analysis of gene expression datasets reveals that Ets1 expression, while low in normal epithelium, increases in pre-malignant and malignant tissues, suggesting a possible role for Ets1 during early tumorigenesis. Strikingly, Kaplan Meier analysis shows that Ets1 expression is associated with both poor survival in HNSCC and high incidence of tumor recurrence. We have performed hierarchical clustering of 33 HNSCC cell lines to show that these cell lines exhibit gene expression profiles that are representative of the four tumor subtypes. To better understand the Ets1-driven molecular processes in EMT high tumors, we have utilized SCC25, a mesenchymal HNSCC cell line to perform global transcriptomic analysis of effects of Ets1 knockdown. Additionally, we have also performed ChIP-seq experiments to identify genome wide transcriptional targets of Ets1 in SCC25. Taken together, these studies have uncovered an Ets1 intrinsic core gene signature that can serve as a valuable tool for further tumor classification and cancer-specific pathway discovery. Interestingly, by cross-referencing with the Cancer Genome Atlas HNSCC RNA-Seq dataset, we demonstrate that Ets1 expression is highly correlated with the majority of the Ets1 signature target genes thus reinforcing a functional relationship. Collectively, our findings not only identify several Ets1 dependent genes that are key to initiating and maintaining EMT in HNSCC, but also unearth novel targets that can be exploited for future therapeutics against this deadly cancer. Citation Format: Christian Gluck, Isha Sethi, Maria Tsompana, Satrajit Sinha. The identification of an Ets1-driven gene signature in head and neck squamous cell carcinoma. [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 A50.

Abstract B20: Malignant transformation initiates a stochastic DNA methylation alteration pattern distinct from that in senescence

Abstract DNA methylation changes are a hallmark of cancers and occur during early tumorigenesis stages. Also, distinct DNA methylation patterns are reported to be characteristic of the anti-tumorigenic process of cell senescence. It is hypothesized that some cells escape senescence and retain the DNA methylation abnormalities manifest in cancers. We now re-visit, in a classic model of tumor progression in human fibroblasts, the relationships between genome-wide DNA methylation patterns associated with senescence, immortalization and transformation. We find that although DNA methylation alterations are a key feature during senescence, these changes are distinct from those we now find associated with malignant transformation in our model and which are classically seen in cancer. Both DNA methylation gains, predominantly at promoter CpG islands, and more balanced losses and gains at other loci, are seen by the time of pre-senescence and remain relatively stable until full senescence is reached. These DNA methylation alterations follow a reproducible pattern amongst independent senescent replicates involving genes in biological processes which positively regulate biosynthesis, gene transcription and macromolecule metabolism. In contrast, during immortalization and malignant transformation, the DNA methylation alterations appear to arise in a stochastic process resulting in heterogeneous methylation patterns amongst independently immortalized and transformed replicates. Gene ontology analysis shows that genes which gain DNA methylation in normally unmethylated promoter CpG island regions in independent transformation replicates enrich for functions involved in developmental regulation. Over the course of progression from immortalization to the transformed state in our model, gene promoter CpG island hypermethylation frequency increases, as does the numbers of associated genes that have decreased gene expression. Thus, a hallmark of the early transformation process is a stochastic accumulation of DNA methylation changes, distinct from those occurring during senescence, that converges to increasingly affect developmental biological processes and diminished transcription of many of the involved genes, which may help drive the process of tumor progression. Citation Format: Wenbing Xie, Yang W. Zhang, Li-Xia Pan, Fillipe Carvlho, Byung-Hak Kang, Limin Xia, Lauren Murphy, Cynthia A. Zahnow, Stephen B. Baylin, Hari Easwaran. Malignant transformation initiates a stochastic DNA methylation alteration pattern distinct from that in senescence. [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 B20.

Overexpressed genes associated with hormones in terminal ductal lobular units identified by global transcriptome analysis: An insight into the anatomic origin of breast cancer.

Although human breast ducts and terminal ductal lobular units (TDLUs) share the same cell types, ample evidence shows that TDLUs are the predominant site for the origin of breast cancer. Yet, there is still limited information concerning the molecular mechanisms. Analysis of transcriptomic profiles in TDLUs may provide insight into early breast tumorigenesis. We compared genome-wide expression profiles of 8 matched sets of breast main duct and TDLU samples, using significance analysis of microarray (SAM) software to screen differentially expressed genes (DEGs) with fold-change >2.0 and q-value <0.05. Moreover, we used Gene Ontology for functional enrichment analysis. We identified 472 DEGs between the two tissue types, and confirmed 17 randomly chosen DEGs by quantitative reverse transcription-PCR (qRT-PCR). Notably, hormone-related pathways were highly enriched in the TDLU samples, including various hormone-related DEGs that are associated with breast carcinogenesis and tumor progression. Oncogenic upregulation in TDLUs indicates a potential inappropriate or excessive response to successive hormone stimulus during the proliferation, differentiation and lactation cycles of the human mammary gland. Imbalanced hormone reactions may finally result in the early onset of neoplastic transformation that occurs mostly in breast TDLUs.

Abstract CN12-03: Epigenetic targets of sulforaphane and epigallocatechin gallate in cancer prevention

Abstract Interest in cancer epigenetics has arisen from the fact that epigenetic processes affect many aspects of tumor formation and progression. The reversibility of epigenetic changes that may arise in early tumorigenesis is an important aspect of their potential in approaches for cancer prevention. Many of the components of the human diet have bioactive effects that lead to reversal of epigenetic aberrations in cancer. We have found that both (-)-epigallocatechin-3-gallate (EGCG) from green tea and sulforaphane (SFN) from cruciferous vegetables are able to down-regulate telomerase in breast cancer cells. This occurs through epigenetic modifications of the promoter region of hTERT, the gene that encodes the catalytic subunit of telomerase. Since telomerase promotes tumor formation and is active in about 90% of cancers, this epigenetic inhibition of its catalytic subunit gene may have considerable potential in breast cancer prevention. Our studies also indicate that these bioactive dietary compounds are able to convert estrogen-receptor (ER)-negative breast cancer cells to ER-positive breast cancer cells and to prevent the formation of highly lethal ER-negative breast cancer. This occurs through epigenetic modifications of the ERalpha gene in response to EGCG and SFN. These findings have significance in that ER-positive cells are amenable to breast cancer prevention or therapy with compounds such as tamoxifen while ER-negative cells confer few preventive or treatment options for patients. Moreover, our studies have also indicated that EGCG and SFN have additive or synergistic epigenetic effects on cancer prevention and that the oral administration of green tea polyphenols and SFN-rich broccoli sprouts inhibits the formation of breast cancer in mice. These results are important because the combination of these dietary products may have potential in lowering their efficacious consumption and increase their utility for breast cancer prevention in humans. Citation Format: Trygve Tollefsbol, Yuanyuan Li. Epigenetic targets of sulforaphane and epigallocatechin gallate in cancer prevention. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr CN12-03.

Targeting of RUNX3 by miR-130a and miR-495 cooperatively increases cell proliferation and tumor angiogenesis in gastric cancer cells.

Mature microRNAs (miRNAs) are 21 to 23 nucleotide noncoding RNA molecules that can downregulate multiple gene expression by mRNA degradation or translational repression. miRNAs are considered to play important roles in cell proliferation, apoptosis, and differentiation during mammalian development. The Runt-related transcription factor 3 (RUNX3) expression and activity are frequently downregulated by various mechanisms in gastric cancer. We have reported that RUNX3 inactivation is crucial for early tumorigenesis. In this study, we investigated the role of miRNAs targeting RUNX3 in early tumorigenesis. miR-130a and miR-495 upregulated under hypoxic conditions that bind to the RUNX3 3'-untranslated region (3'-UTR) were identified in gastric cancer cells by using microarray analysis and bioinformatics programs. Combination of miR-130a and miR-495 inhibited RUNX3 expression at the protein level, but not at the mRNA level. miR-130a and miR-495 significantly inhibited the RUNX3-3'UTR-luciferase activity. Combination of miR-130a and miR-495 significantly decreased apoptosis determined by Annexin V-FITC/propidium iodide staining and flow cytometric analysis, and the expression of Bim in SNU484 gastric cancer cells. In addition, p21 and Bim, RUNX3 target genes, were completely downregulated by the combination of miR-130a and miR-495. Using matrigel plug assay, we found that antagomiRs specific for miR-130a and miR-495 significantly reduced angiogenesis in vivo. In conclusion, targeting miR-130a and miR-495 could be a potential therapeutics to recover RUNX3 expression under hypoxic conditions and in early tumorigenic progression.

MicroRNA-200 promotes lung cancer cell growth through FOG2-independent AKT activation.

MicroRNA-200 (miR-200) has emerged as a regulator of the PI3K/AKT pathway and cancer cell growth. It was reported that miR-200 can activate PI3K/AKT by targeting FOG2 (friend of GATA 2), which directly binds to the p85α regulatory subunit of PI3K. We found that miR-200 was elevated in early stage lung adenocarcinomas when compared with normal lung tissues, and the expression of miR-200 promoted the tumor spheroid growth of lung adenocarcinoma cells. We show that AKT activation was essential for such oncogenic action of miR-200. However, depletion of FOG2 had little effect on AKT activation. By performing a reverse-phase protein array, we found that miR-200 not only activated AKT but also concomitantly inactivated S6K and increased IRS-1, an S6K substrate that is increased on S6K inactivation. Depletion of IRS-1 partially inhibited the miR-200-dependent AKT activation. Taken together, our results suggest that miR-200 may activate AKT in lung adenocarcinoma cells through a FOG2-independent mechanism involving IRS-1. Our findings also provide evidence that increased miR-200 expression may contribute to early lung tumorigenesis and that AKT inhibitors may be useful for the treatment of miR-200-dependent tumor cell growth.

Abstract POSTER-CTRL-1204: Novel strategies for targeting high-grade serous ovarian cancer

Abstract High-grade serous ovarian carcinoma (HGSC) presents significant clinical and therapeutic challenges. Emerging data from The Cancer Genome Atlas (TCGA) have identified a number of new genetic aberrations but their functional significance and the role they play in early tumorigenesis remains to be determined. In addition, identifying the key genetic lesions responsible for tumor initiation and the specific cell of origin is a priority for ovarian cancer research. Although the traditional model of carcinogenesis has focused on the ovary as a tumor initiation site, recent preclinical and clinical studies have suggested that there are additional sites of origin outside the ovary, namely fallopian tubal secretory epithelial cells (FTSECs). This new model of tumorigenesis has further identified novel entities in the fallopian tube epithelium that represent precursors to invasive carcinoma – the TP53 signature and serous tubal intraepithelial carcinoma (STIC). Identifying and characterizing the events leading to the development of TP53 signatures and STICs, the critical steps in their transition to malignancy, and novel methods for detecting this transition prior to cancer development are critical for cancer prevention and development of personalized approaches to therapy. We have successfully developed genetic models of de novo HGSC that originate in FTSECs and mimic the key genetic alterations and precursor lesions characteristic of human invasive ovarian cancer. The murine tumors accurately recapitulate the histologic, immunophenotypic (CK-8, STMN1, PAX2, P53, Ki-67, WT1, and CA-125), and genomic alterations observed in human HGSC, thus providing a compelling argument for serous carcinogenesis originating in the FTSEC. Our models serve as a proof-of-concept that high-grade serous “ovarian” cancer can arise from the FTSEC and progress to metastatic disease via pre-invasive lesions, namely the STICs. We are currently using the Pax8-driven system to interrogate the role of TCGA genes that have been identified as being important for serous tumorigenesis. Successful completion of our studies will demonstrate that the fallopian tubal secretory epithelial cell is indeed a cell-of-origin for ovarian and pelvic serous carcinomas in vivo when using the most common key TCGA-based tumor drivers for BRCA and non-BRCA tumors, respectively. The first goal of our research is be to produce valid models for HGSC tumors. The second is to identify unique characteristics of early serous carcinogenesis in the tube that can be exploited for early detection. The third is to determine the causes of the earliest events preceding malignancy, which will enable more efficient methods of diagnostic imaging and cancer prevention with a focus on the distal fallopian tube. Furthermore, by using a combination of murine model studies and epidemiological data from patients, it will be important to determine if premenopausal women with BRCA mutations can be offered risk-reduction surgery in a multi-step procedure without undergoing surgical menopause and loss of fertility in their younger years. Citation Format: Ruth Perets, Gregory A Wyant, Anders W Ohman, Sunita R Setlur, Christopher P. Crum, Ronny Drapkin, Daniela M. Dinulescu. Novel strategies for targeting high-grade serous ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-CTRL-1204.

Abstract POSTER-TECH-1122: Mass spectrometric imaging of ovarian cancer mouse model tissue: searching for high-grade serous carcinoma biomarkers

Abstract High-grade serous carcinoma (HGSC) is the most common and deadliest ovarian cancer responsible for 90% of all ovarian cancer deaths. Studying the origin and early progression of this deadly cancer is critical in identifying biomarkers for early detection of the disease. Unfortunately, in the case of HGSC, it is virtually impossible to obtain early-stage human HGSC tissue samples because there is no diagnostic method capable of detecting early forms of HGSC. The recent breakthrough of an animal model (Dicer-Pten double-knockout mice) provides an invaluable opportunity to finally investigate the early tumor microenvironment. The model exhibits remarkable similarities to human HGSC, allowing greater insight into the biochemistry associated with nascent neoplastic changes within ovarian and fallopian tube tissues. An important aspect of determining potential biomarkers is to correlate any detected chemical changes with observed histological changes. To visualize the spatial distribution of chemical changes, tissue-based mass spectrometric imaging (MSI) can be employed as an unbiased way of visualizing the changes of proteins, metabolites, or both in cancer tissues. MSI is particularly useful for identifying molecules uniquely elevated or lowered in early tumor tissues, in which cell-type specific changes are expected to occur due to intratumoral microenvironment heterogeneity. In this study, two complementary MSI techniques – matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) mass spectrometry – are applied to thin tissue sections of Dicer-Pten mutant mouse ovaries and fallopian tubes. Several sulfatide lipids d18:1/C16:0, d18:1/C24:1 and d18:1/C24:0 have been implicated as potential biomarkers in human ovarian cancer. These metabolites, among others, are being included in a thorough MSI experiment to visualize molecular changes in multiple cell types in the microenvironment during early tumor progression. In addition to providing information about the early molecular changes taking place, the knowledge gained from this study will also help understand which specific cell types in the fallopian tube are more prone to early tumorigenesis. (Grant support: Marsha Rivkin Ovarian Cancer Challenge Grant) Citation Format: Martin R. L. Paine, Rachel V. Bennett, Jaeyeon Kim, M. Cameron Sullards, Martin M. Matzuk, Facundo M. Fernández. Mass spectrometric imaging of ovarian cancer mouse model tissue: searching for high-grade serous carcinoma biomarkers [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1122.

Niche appropriation by Drosophila intestinal stem celltumours.

Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. We investigated Drosophila intestinal stem cell (ISC) tumors generated by suppressing Notch (N) signaling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumor initiation and an autocrine EGFR ligand, Spitz, during early tumor growth. Upon achieving a critical mass these tumors displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumor growth without the need for secondary mutations in growth signaling pathways. The appropriation of niche signaling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis.