Early Stages Of Tumorigenesis Research Articles

PO-477 A novel role for nanog as an early cancer risk marker in patients with laryngeal precancerous lesions

IntroductionNANOG is a transcription factor involved in embryonic pluripotency maintenance. It has also been shown that it has a mitogenic function in adult epithelial tissues. Overexpression of NANOG in mice causes hyperplasia, increased proliferation and aneuploidy. In humans, it has been found to be frequently aberrantly expressed in a variety of cancers, including head and neck squamous cell carcinomas (HNSCC). This overexpression has been associated with chemoresistance, epithelial to mesenchymal transformation (EMT) and poor clinical outcome. Even if there is enough evidence supporting the oncogenic role of NANOG, its role in the early stages of tumorigenesis in HNSCC has not been assessed. This study investigates for the first time the role of NANOG expression in early stages of laryngeal tumourigenesis and its potential utility as cancer risk marker.Material and methodsNANOG protein expression was evaluated by immunohistochemistry in a group of 82 patients from Asturias with laryngeal precancerous lesions, and correlated with clinicopathological parameters and laryngeal cancer risk. Results were validated in an independent cohort of 86 patients from Barcelona.Results and discussionsNANOG expression was detected in the cytoplasm by immunohistochemistry in 49 (60%) of 82 laryngeal dysplasias, whereas expression was negligible in patient-matched normal epithelia. Strong NANOG expression was found in 22 (27%) lesions. 24 of the patients (29%) developed an invasive carcinoma on the site of the lesion in the following months. No significant differences between the group that developed cancer and the one that did not were found concerning age (p=0.703), smoking (p=0.316) or histopathological diagnosis (p=0.748). Cytoplasmic expression of NANOG showed the most robust association with laryngeal cancer risk (p=0.007). Patients with a strong expression of cytoplasmic NANOG had a higher risk of getting laryngeal cancer (HR=1.826, p=0.003). Similar trends were obtained using the validation cohort. Our findings show NANOG is frequently overexpressed in laryngeal dysplasias but not in adjacent normal epithelia. Strong expression of cytoplasmic NANOG significantly correlated with a higher cancer incidence, showing a superior predictive power than histological classification, which is the current gold standard to determine cancer risk.ConclusionTogether our studies uncover a novel role for NANOG expression in laryngeal tumourigenesis, and its unprecedented application as biomarker for cancer risk assessment.

Abstract 3081: Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer

Abstract Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by germline mutations in the DNA mismatch repair (MMR) genes resulting in an increased risk of colorectal cancer (CRC) and other malignancies. Those germline defects with other genetic and epigenetic changes accelerate multistep tumorigenesis. However, early events leading to polyp formation and the timing and order of the molecular “hits” remain unknown. Studies on sporadic CRC have indicated that promoter hypermethylation leading to gene silencing can act as an alternative mechanism to mutations at early stages of tumorigenesis, but its importance in hereditary CRC is obscure. In this study we aimed to define 1) methylation changes that occur at different stages of LS-associated colorectal tumor progression, and 2) somatic mutations that occur in adenomas comparing the status of low- and high-grade dysplasia and MMR deficiency (MMR-D) and proficiency (MMR-P). Colorectal biopsies including normal mucosae, adenomas and carcinomas were prospectively collected from 104 LS patients during colonoscopy surveillance, supplemented with retrospective tumor specimens from 56 patients. Promoter methylation was analyzed using the methylation-specific multiplex ligation-dependent probe amplification test (MS-MLPA) including selected tumor suppressor genes (TSGs) associated with early colon oncogenesis. Hypermethylation tendency was evaluated based on the frequency of CpG island methylator phenotype (CIMP) according to the methylation status of eight established CIMP marker genes. LINE-1 methylation was studied as a surrogate marker for global hypomethylation. Immunohistochemistry was used to detect MMR protein expression in neoplastic lesions. As an ongoing study mutation status of adenomas are studied using the Ion AmpliSeqTM Colon and Lung Cancer Panel targeting mutational hot spots in 22 genes. Results indicate that the expression of the MMR protein corresponding to the gene mutated in the germline decreases along with dysplasia but occurs as a relatively late event in the tumor progression, suggesting the presence of other somatic events that drive neoplastic transformation. Taken together, methylation of TSGs and frequency of CIMP increased and LINE-1 methylation decreased in adenomas and carcinomas along with dysplasia. However, a significant increase of methylation in some genes was detected already in adenomas with low-grade dysplasia compared to normal mucosae. In addition, a proportion of low-grade adenomas could already be classified CIMP positive. When comparing MMR-P and MMR-D adenomas, LINE-1 methylation decreased along the loss of MMR protein expression, and interestingly, higher methylation of SFRP1 was observed in MMR-P adenomas compared to MMR-D cases and normal mucosae. These findings emphasize the importance and early appearance of epigenetic changes in LS-associated tumorigenesis. Citation Format: Satu Mäki-Nevala, Satu Valo, Ari Ristimäki, Laura Renkonen-Sinisalo, Anna Lepistö, Jukka-Pekka Mecklin, Päivi Peltomäki. Methylation changes and somatic mutations as early events in Lynch syndrome-associated colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3081.

Abstract 4181: Simultaneous inhibition of the Wnt/B-catenin and Ras pathways by KY1022 effectively suppresses tumor initiation and progression of colorectal cancer

Abstract APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both B-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Targeting these signaling pathways may therefore be an ideal strategy for treating mCRC; however, therapeutics targeting both of these pathways have not been developed because the mechanisms regulating these pathways are not well understood. Based on our recent identification of the B-catenin destruction complex-mediated Ras protein stability mechanism, we screened a small-molecule library to identify compounds that destabilize both B-catenin and Ras proteins via inhibition of the Wnt/B-catenin pathway. Among the effective small molecules destabilizing both B-catenin and Ras proteins via inhibition of the Wnt/β-catenin pathway, we characterized KY1022, a small molecule that most effectively inhibits EMT, an initial process of metastasis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. Our results suggest that a small molecular approach degrading both B-catenin and Ras via inhibition of the Wnt/B-catenin signaling would be an ideal strategy for treatment of CRC including mCRC. Citation Format: Eun Ji Ro, Jeong-Ha Hwang, Yong-Hee Cho, Kang-Yell Choi. Simultaneous inhibition of the Wnt/B-catenin and Ras pathways by KY1022 effectively suppresses tumor initiation and progression of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4181.

Immunohistochemical Expression of Cortactin and Focal Adhesion Kinase Predicts Recurrence Risk and Laryngeal Cancer Risk Beyond Histologic Grading.

Background: Cortactin (CTTN) and the focal adhesion kinase (FAK) are two major candidate genes to, respectively, drive 11q13- and 8q24-associated aggressive behavior in various cancers. Recent evidence uncovered their clinical relevance in early stages of tumorigenesis as promising biomarkers for cancer risk assessment.Methods: Using a multicenter validation study, CTTN and FAK expression was evaluated by immunohistochemistry (IHC) in a cohort of 109 patients with laryngeal precancerous lesions, and correlated with clinicopathologic parameters and laryngeal cancer risk. The pathophysiologic role of CTTN and FAK was further investigated using functional studies in cellular models.Results: Positive CTTN and FAK expression (scores 2 and 3) was detected in 49 (41%) and 35 (32%) laryngeal dysplasias, respectively. Univariate Cox analysis showed that CTTN and FAK expression but not histologic grading was significantly associated with both recurrence risk and laryngeal cancer risk. Patients carrying strong CTTN- or FAK-expressing lesions (score 3) experienced the highest laryngeal cancer incidence (log-rank P < 0.001). In multivariate stepwise analysis, FAK expression [HR = 13.91; 95% CI, 4.82-40.15; P < 0.001] and alcohol consumption (HR = 2.22; 95% confidence interval, 1.17-4.20; P = 0.014) were significant independent predictors of laryngeal cancer development. Targeting FAK by either RNAi or pharmacologic inhibitors effectively blocked cell growth, colony formation, and invasion into 3D collagen matrices.Conclusions: CTTN and FAK emerge as powerful predictors of laryngeal cancer risk and recurrence risk beyond histologic grading.Impact: Our work supports the applicability of IHC CTTN and FAK as complementary markers for risk stratification in patients with laryngeal precancerous lesions. Cancer Epidemiol Biomarkers Prev; 27(7); 805-13. ©2018 AACR.

The contributions of cancer cell metabolism to metastasis.

ABSTRACTMetastasis remains the leading cause of cancer-related deaths worldwide, and our inability to identify the tumour cells that colonize distant sites hampers the development of effective anti-metastatic therapies. However, with recent research advances we are beginning to distinguish metastasis-initiating cells from their non-metastatic counterparts. Importantly, advances in genome sequencing indicate that the acquisition of metastatic competency does not involve the progressive accumulation of driver mutations; moreover, in the early stages of tumorigenesis, cancer cells harbour combinations of driver mutations that endow them with metastatic competency. Novel findings highlight that cells can disseminate to distant sites early during primary tumour growth, remaining dormant and untreatable for long periods before metastasizing. Thus, metastatic cells must require local and systemic influences to generate metastases. This hypothesis suggests that factors derived from our lifestyle, such as our diet, exert a strong influence on tumour progression, and that such factors could be modulated if understood. Here, we summarize the recent findings on how specific metabolic cues modulate the behaviour of metastatic cells and how they influence the genome and epigenome of metastatic cells. We also discuss how crosstalk between metabolism and the epigenome can be harnessed to develop new anti-metastatic therapies.

Ectopic epithelial cell clusters in salmonid intestine are associated with inflammation.

An epizootic incidence of intestinal adenocarcinomas was reported in brood fish of Atlantic salmon (Salmo salar L.) in 2009. The condition was associated with a specific diet inducing enteritis and morphological changes. Here, two field trials of fish up to slaughter size were initiated. In Trial 1, two different feed recipes were used. Feed I was predominantly based on marine ingredients, whereas plant ingredients were limited to soy protein concentrate and wheat. Feed II was lower in fishmeal and without soya protein, which was substituted with plant proteins from other sources. In Trial 2, a commercial feed (Feed III) was included. No macroscopic tumours were observed in 300 fish (Trial 1). At the end of both trials, samples from five different segments of the gastrointestinal tract of a total of 39 fish were investigated with morphological methods. Here, we show the presence of ectopic proliferating epithelial cells only occurring in inflamed intestine and predominantly in the second segment of the mid-intestine. Presence of ectopic epithelial cells in submucosal inflammatory foci may indicate early stages in tumorigenesis, but other possibilities such as proliferative enteric disorders cannot be excluded. Together with inflammation, carcinogenesis should be a focus of investigation in future feed trials.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC’s orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

The inflammatory microenvironment that promotes gastrointestinal cancer development and invasion.

Accumulating evidence has indicated that the inflammatory response is important for tumor promotion. However, the mechanisms underlying the induction of the inflammatory response in cancer tissues and how it promotes tumorigenesis remain poorly understood. We constructed several mouse models that develop inflammation-associated gastric and intestinal tumors and examined the in vivo mechanisms of tumorigenesis. Of note, the activation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway and Toll-like receptor (TLR)/MyD88 signaling cooperatively induced the generation of an inflammatory microenvironment, which is required for early-stage tumorigenesis. The inflammatory response in the stroma induces TNF-α signaling in tumor cells, and the NOX1/ROS signaling pathway is activated downstream. In addition, the inflammatory pathway induces the expression of TLR2 in tumor epithelial cells. Both the NOX1/ROS and TLR2 pathways in tumor cells contribute to the acquisition and maintenance of stemness, which is an important tumor-promoting mechanism stimulated by inflammation. We also found that inflammation promotes malignant processes, like submucosal invasion, of TGF-β signaling-suppressed tumor cells through the activation of MMP2 protease. Moreover, we showed that mutant p53 induces innate immune and inflammatory signaling in the tumor stroma by a gain-of-function mechanism of mutant p53, which may explain the "cancer-induced inflammation" mechanism. These results indicate that the regulation of the inflammatory microenvironment via the inhibition of the COX-2/PGE2 and TLR/MyD88 pathways in combination will be an effective preventive or therapeutic strategy against gastrointestinal cancer development and malignant progression, especially those carrying p53 gain-of-function mutations.

An Anti-tumorigenic Role of the Warburg Effect at Emergence of Transformed Cells

The Warburg effect is one of the hallmarks of cancer cells, characterized by enhanced aerobic glycolysis. Despite intense research efforts, its functional relevance or biological significance to facilitate tumor progression is still debatable. Hence the question persists when and how the Warburg effect contributes to carcinogenesis. Especially, the role of metabolic changes at a very early stage of tumorigenesis has received relatively little attention, and how aerobic glycolysis impacts tumor incidence remains largely unknown. Here we discuss a novel paradigm for the effect of the Warburg effect that provides a suppressive role in oncogenesis.Key words: Warburg effect, aerobic glycolysis, cell competition, EDAC.

Virotherapy of the Malignant U87 Human Glioblastoma in the Orthotopic Xenotransplantation Mouse SCID Model.

The possibility of glioblastoma virotherapy at intravenous injection of the LIVP-GFP recombinant virus was studied in experimental model of orthotopic xenotransplantation of human glioblastoma cell line U87 to SCID laboratory mice. The LIVP-GFP recombinant virus deficient for thymidine kinase exhibited a significantly greater oncolytic capacity than the original LIVP virus, and an intravenous injection of LIVP-GFP at the early stages of tumorigenesis in mouse brain in most cases resulted in the lysis of the tumor.

Cilia loss sensitizes cells to transformation by activating the mevalonate pathway.

Although cilia loss and cell transformation are frequently observed in the early stage of tumorigenesis, the roles of cilia in cell transformation are unknown. In this study, disrupted ciliogenesis was observed in cancer cells and pancreatic cancer tissues, which facilitated oncogene-induced transformation of normal pancreatic cells (HPDE6C7) and NIH3T3 cells through activating the mevalonate (MVA) pathway. Disruption of ciliogenesis up-regulated MVA enzymes through β catenin-T cell factor (TCF) signaling, which synchronized with sterol regulatory element binding transcription factor 2 (SREBP2), and the regulation of MVA by β-catenin-TCF signaling was recapitulated in a mouse model of pancreatic ductal adenocarcinoma (PDAC) and human PDAC samples. Moreover, disruption of ciliogenesis by depleting Tg737 dramatically promoted tumorigenesis in the PDAC mouse model, driven by KrasG12D , which was inhibited by statin, an inhibitor of the MVA pathway. Collectively, this study emphasizes the crucial roles of cilia in governing the early steps of the transformation by activating the MVA pathway, suggesting that statin has therapeutic potential for pancreatic cancer treatment.

Telomere Shortening in Hematological Malignancies with Tetraploidization-A Mechanism for Chromosomal Instability?

Aneuploidy, the presence of an abnormal number of chromosomes in a cell, is one of the most obvious differences between normal and cancer cells. There is, however, debate on how aneuploid cells arise and whether or not they are a cause or a consequence of tumorigenesis. Further, it is important to distinguish aneuploidy (the “state” of the karyotype) from chromosomal instability (CIN; the “rate” of karyotypic change). Although CIN leads to aneuploidy, not all aneuploid cells exhibit CIN. One proposed route to aneuploid cells is through an unstable tetraploid intermediate because tetraploidy promotes chromosomal aberrations and tumorigenesis. Tetraploidy or near-tetraploidy (T/NT) (81–103 chromosomes) karyotypes with or without additional structural abnormalities have been reported in acute leukemia, T-cell and B-cell lymphomas, and solid tumors. In solid tumors it has been shown that tetraploidization can occur in response to loss of telomere protection in the early stages of tumorigenesis in colon cancer, Barrett’s esophagus, and breast and cervical cancers. In hematological malignancies T/NT karyotypes are rare and the role of telomere dysfunction for the induction of tetraploidization is less well characterized. To further our understanding of possible telomere dysfunction as a mechanism for tetrapolydization in hematological cancers we here characterized the chromosomal complement and measured the telomere content by interphase nuclei quantitative fluorescence in situ hybridization (iQFISH) in seven hematological cancer patients with T/NT karyotypes, and after cytogenetic remission. The patients were identified after a search in our local cytogenetic registry in the 5-year period between June 2012 and May 2017 among more than 12,000 analyzed adult patients in this period. One advantage of measuring telomere content by iQFISH is that it is a single-cell analysis so that the telomere content can be distinguished between normal karyotype cells and cells with T/NT karyotypes. We find that the telomeres are particularly short in cells with T/NT karyotypes as compared with normal cells, and in T/NT karyotypes harboring additional chromosomal aberrations as well. These findings suggest that telomere dysfunction in hematological malignancies may be a mechanism for tetraploidization and CIN.

Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neutrophils.

Bone marrow-derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients (n = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.

A bona fide portrayal of tumor growth

Cancer Bone has a well-established role in advanced cancer. It provides a supportive microenvironment for the growth of metastatic cells that escape the primary tumor, which ultimately leads to loss of bone mass. Engblom et al. show that bone may also contribute to early-stage tumorigenesis through a mechanism that leads to an increase in bone mass (see the Perspective by Zhang and Lyden). In mouse models of lung adenocarcinoma, primary tumor cells remotely activated bone-resident cells called osteoblasts, which have a bone-building function. The activated osteoblasts in turn triggered production of a certain type of neutrophil that infiltrates the primary tumor and promotes its growth. Patients with early-stage lung cancer were also found to have an increase in bone density, consistent with the findings in mice. Science , this issue p. [eaal5081][1]; see also p. [1127][2] [1]: /lookup/doi/10.1126/science.aal5081 [2]: /lookup/doi/10.1126/science.aar2640

Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion.

Mammography screening has increased the detection of early pre-invasive breast cancers, termed ductal carcinoma in situ (DCIS), increasing the urgency of identifying molecular regulators of invasion as prognostic markers to predict local relapse. Using the MMTV-PyMT breast cancer model and pharmacological protease inhibitors, we reveal that cysteine cathepsins have important roles in early-stage tumorigenesis. To characterize the cell-specific roles of cathepsins in early invasion, we developed a DCIS-like model, incorporating an immortalized myoepithelial cell line (N1ME) that restrained tumor cell invasion in 3D culture. Using this model, we identified an important myoepithelial-specific function of the cysteine cathepsin inhibitor stefin A in suppressing invasion, whereby targeted stefin A loss in N1ME cells blocked myoepithelial-induced suppression of breast cancer cell invasion. Enhanced invasion observed in 3D cultures with N1ME stefin A-low cells was reliant on cathepsin B activation, as addition of the small molecule inhibitor CA-074 rescued the DCIS-like non-invasive phenotype. Importantly, we confirmed that stefin A was indeed abundant in myoepithelial cells in breast tissue. Use of a 138-patient cohort confirmed that myoepithelial stefin A (cystatin A) is abundant in normal breast ducts and low-grade DCIS but reduced in high-grade DCIS, supporting myoepithelial stefin A as a candidate marker of lower risk of invasive relapse. We have therefore identified myoepithelial cell stefin A as a suppressor of early tumor invasion and a candidate marker to distinguish patients who are at low risk of developing invasive breast cancer, and can therefore be spared further treatment. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Trichostatin A preferentially reverses the upregulation of gene-expression levels induced by gain of chromosome 7 in colorectal cancer cell lines.

Epithelial cancers are defined by a tumor-specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast, and pancreatic cancer. These ploidy-driven transcriptional deregulations are characterized by low-level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggest that aneuploidy-dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large-scale gene-expression changes. Here we assessed if TSA could disrupt the aneuploidy-driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD-1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy-induced gene-expression changes on chromosome 7. Among the TSA affected genes on chromosome 7, we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy-driven gene-expression changes.

DNA methylation aberrancies delineate clinically distinct subsets of colorectal cancer and provide novel targets for epigenetic therapies.

Colorectal cancer (CRC) is a worldwide health concern with respect to both incidence and mortality, and as a result, CRC tumorigenesis, progression and metastasis have been heavily studied, especially with respect to identifying genetic, epigenetic, transcriptomic and proteomic profiles of disease. DNA methylation alterations are hallmarks of CRC, and epigenetic driver genes have been identified that are thought to be involved in early stages of tumorigenesis. Moreover, distinct CRC patient subgroups are organized based on DNA methylation profiles. CRC tumors displaying CpG island methylator phenotypes (CIMPs), defined as DNA hypermethylation at specific CpG islands in subsets of tumors, show high concordance with specific genetic alterations, disease risk factors and patient outcome. This review details the DNA methylation alterations in CRC, the significance of CIMP status, the development of treatments based on specific molecular profiles and the application of epigenetic therapies for CRC patient treatment.

NF-κB regulates GDF-15 to suppress macrophage surveillance during early tumor development.

Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-β-activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.

Abstract 2915: The timing of mutational burst events impact the growth of tumors in the colon

Abstract Basic and clinical scientists believe that benign polyps in the colon progress to cancers through the slow, stepwise accumulation of mutations. Interestingly, only a small percentage of all tumors progress, whereas a significant number remain static in size, regress, or resolve completely. The mechanisms underlying these differential fates are unknown, and mechanisms of tumor evolution during this premalignant phase are still under investigation and continued debate. We previously reported that sub-clonal diversity arises early in small human adenomas and contributes to the growth of tumors in the colon. An emerging hypothesis is that colorectal tumors form and progress via a process of punctuated equilibrium, where multiple copy number alterations and mutational events happen simultaneously in a burst-like fashion. In this study, we prospectively test this concept using a mouse model in which tumor induction is spatially and temporally controlled via a non-surgical delivery of adenovirus expressing CRE recombinase and a temporally controlled mutational burst via administration of the carcinogen Azoxymethane. Tumors are induced at a similar rate regardless the timing of the burst relative to tumor induction with a mean tumor incidence of 62% at three weeks post induction. However, tumor growth may be affected by the timing of the burst. Animals that had a mutational burst event prior to tumor induction or those that had a late mutational burst event had a lower average in vivo growth rate compared to controls and those with an early burst event, but the average in vivo tumor size was comparable across all groups. Taken together, these preliminary data provide evidence that the timing of a mutational burst event contributes to tumor growth. This prospective experiment is being extended through computer modeling and statistical inference to compare in silico mutational landscapes to a cohort of colon polyps removed from patients at normal screening. The findings will provide new insights into the earliest stages of tumorigenesis. Citation Format: Chelsie K. Sievers, Tien N. Vo, Perry J. Pickhardt, Bryan D. Pooler, Kristina A. Matkowskyj, Dawn M. Albrecht, Quincy Rosemarie, Michael A. Newton, Richard B. Halberg. The timing of mutational burst events impact the growth of tumors in the colon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2915. doi:10.1158/1538-7445.AM2017-2915

Abstract 5748: Roles of miR29c-ID2 axis in tumor initiating cells of breast ductal carcinoma in situ

Abstract Ductal carcinoma in situ (DCIS) is an early-stage form of breast cancer that accounts for over 20% of all breast cancers in the US. Therefore, prevention of DCIS is a major goal for reducing breast cancer incidence. However, the underlying molecular and pathological mechanisms of DCIS remain poorly understood. It is believed that tumor-initiating cells are formed at a very early stage of tumorigenesis, which then leads to the progression of DCIS. Our recent work that focused on tumor-initiating cells of DCIS revealed that the expression of miR29c in tumor-initiating cells was significantly down-regulated in DCIS cells compared to that in normal cells. Furthermore, we found that the ID2 gene is one of the targets of miR29c in tumor-initiating cells and that miR29c suppresses the ID2 gene by directly binding to the 3’-UTR sequence. ID2 is a well-known dedifferentiation factor in embryonic development, and the ID family is involved in progression of cancer by affecting cell differentiation and proliferation. We also found that ectopic expression of ID2 in premalignant cells accelerated tumor growth and promoted self-renewal of tumor-initiating cells in DCIS, suggesting that ID2 also controls cancer cell dedifferentiation and promotes tumor-initiating cells formation. These results indicate that the miR29c-ID2 axis plays a critical role in self-renewal of DICS in vivo and that ID2 is a potential biomarker for tumor-initiating cells in DCIS. ID2 is thought to sequester E-protein transcription factors and thus regulates a wide range of gene expression. Our data suggest that ID2 upregulates expression of several cancers stem cell genes including SOX2, which is a well- known self-renewal regulator of stem cells. ID2 also upregulates the fatty acid synthase gene (FASN), which promotes cancer cell proliferation and survival. We also found that FASN expression was significantly suppressed by pterostilbene, indicating potential utility of this natural compound for preventing DCIS formation. Taken together, these results suggest that miR29c-ID2 functions as master regulators in forming DCIS. ID2 promotes self-renewal of tumor-initiating cells by regulating expression of the SOX2 and FASN genes. Note: This abstract was not presented at the meeting. Citation Format: Yin Liu, Puspa R. Pandey, Sambad Sharma, Kerui Wu, Fei Xing, Kounosuke Watabe. Roles of miR29c-ID2 axis in tumor initiating cells of breast ductal carcinoma in situ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5748. doi:10.1158/1538-7445.AM2017-5748