Early Stages Of Carcinogenesis Research Articles

Abstract A039: A sensitive tool for the detection of RNA modifications in blood samples

Abstract The epitranscriptome is the set of chemical modifications applied naturally to RNA to regulate seemingly all aspects of its function, including splicing, translation initiation and fidelity, trafficking, RNA folding and stability, and transcript lifetime. Detecting RNA in blood reliably continues to be a challenge due to its labile nature, and detecting RNA modifications is an even greater challenge. For this reason, current liquid biopsy approaches concentrate on determining the mutational burden in cell-free DNA and do not harness the potential of cfRNA. In contrast to cfDNA, cfRNA is a biomarker for dynamic events that are known to occur at early stages of carcinogenesis, such as alterations to transcriptional profiles and other aspects of the regulatory state of cells. In many RNA species, the modification state is linked to the RNA lifecycle, thus providing important insights into cell state. For example, pre-miRNA is modified before miRNA maturation, tRNA cleavage is initiated by changes to the modification pattern and mRNA is decapped for storage in stress granules. AlidaBio is introducing the EpiPlex platform, an assay platform with an integrated bioinformatics solution that is designed for ultra-low RNA input and has been tested with blood samples. In addition to its sensitivity, the EpiPlex platform has the advantage of detecting multiple RNA modifications in the same reaction, enabling users to elucidate how RNA modifications act in concert to fine-tune RNA function. The approach uses targeted, multiplexed barcoding to reveal co-localization of modifications on the same molecule and to provide semi-quantitative data on mod abundance. This sensitive, multiplexed target detection has the potential to advance the field of RNA liquid biopsy with new biomarker discovery. Citation Format: Andrew Price, Zachary Miles, Byron Purse, Gudrun Stengel. A sensitive tool for the detection of RNA modifications in blood samples [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A039.

From Inception to Malignancy: the Co-evolution of Pancreatic Cancer and Its Immunosuppressive Microenvironment.

Published in Cancer Research in 2007, Clark and colleagues first introduced the concept that the immune microenvironment evolves in lockstep with the progression of pancreatic cancer. Leveraging genetically engineered mouse models of the disease that were described a few years earlier, Clark and colleagues used a combination of approaches to describe the dynamics of immune evolution in precursor lesions all the way to overt malignancy. They discovered that immunosuppression is established at the earliest stages of carcinogenesis. Here, we discuss their findings, how they led to a wealth of functional work, and how they have been expanded upon since the advent of -omics technologies. See related article by Clark and colleagues, Cancer Res 2007;67:9518-27.

Oat Beta-Glucan as a Metabolic Regulator in Early Stage of Colorectal Cancer-A Model Study on Azoxymethane-Treated Rats.

Factors that reduce the risk of developing colorectal cancer include biologically active substances. In our previous research, we demonstrated the anti-inflammatory, immunomodulatory, and antioxidant effects of oat beta-glucans in gastrointestinal disease models. The aim of this study was to investigate the effect of an 8-week consumption of a diet supplemented with low-molar-mass oat beta-glucan in two doses on the antioxidant potential, inflammatory parameters, and colonic metabolomic profile in azoxymethane(AOM)-induced early-stage colorectal cancer in the large intestine wall of rats. The results showed a statistically significant effect of AOM leading to the development of neoplastic changes in the colon. Consumption of beta-glucans induced changes in colonic antioxidant potential parameters, including an increase in total antioxidant status, a decrease in the superoxide dismutase (SOD) activity, and a reduction in thiobarbituric acid reactive substance (TBARS) concentration. In addition, beta-glucans decreased the levels of pro-inflammatory interleukins (IL-1α, IL-1β, IL-12) and C-reactive protein (CRP) while increasing the concentration of IL-10. Metabolomic studies confirmed the efficacy of oat beta-glucans in the AOM-induced early-stage colon cancer model by increasing the levels of metabolites involved in metabolic pathways, such as amino acids, purine, biotin, and folate. In conclusion, these results suggest a wide range of mechanisms involved in altering colonic metabolism during the early stage of carcinogenesis and a strong influence of low-molar-mass oat beta-glucan, administered as dietary supplement, in modulating these mechanisms.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis

Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Abstract A109: Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis

Abstract Introduction The major driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods To elucidate mechanisms responsible for the accelerated cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Differentially regulated genes were validated by multiplex RNAscope and immunohistochemistry staining and functionally studied in pancreatic cancer cell lines. Results Single-cell ATAC-seq proved a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. Notably, acinar cells of KrasG12D;Pdx1f/f animals as well as a proportion of metaplastic lesions in both, KrasG12D and KrasG12D;Pdx1f/f mice, showed elevated accessibility and expression of the Ror2 (Receptor Tyrosine Kinase Like Orphan Receptor 2) gene. As a receptor tyrosine kinase, Ror2 controls noncanonical Wnt signaling and other essential signaling pathways, such as PI3K/AKT or Ras-MAPK. Genetic ablation of Ror2 in a mouse model of pancreatic neoplasia resulted in a shift in ADM cell identity, enriching ADM lesions with a senescent duct cell phenotype. In PDAC, ROR2 expression correlates with the more aggressive basal-like subtype. Overexpression of ROR2 in pancreatic cancer cell lines with a classical differentiation induced epithelial-to-mesenchymal transition, characterized by the downregulation of multiple epithelial markers and upregulation of mesenchymal genes. Knockout of ROR2 in pancreatic cancer cells significantly decreased cell proliferation. Conclusions Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 leads to vast alterations of acinar cell identity. We identified the receptor kinase Ror2 as a regulator of pancreatic cancer initiation and driver of pancreatic cancer cell aggressiveness. Citation Format: Simone Benitz, Malak Nasser, Alexander Steep, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Erick Davis, Hui-Ju Wen, Daniel Long, Michaela Louw, Samuel Zwernik, Donald Rempinski, Jacee Moore, Daniel Salas-Escabillas, Thomas Metzler, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A109.

Microbial metabolites are involved in tumorigenesis and development by regulating immune responses.

The human microbiota is symbiotic with the host and can create a variety of metabolites. Under normal conditions, microbial metabolites can regulate host immune function and eliminate abnormal cells in a timely manner. However, when metabolite production is abnormal, the host immune system might be unable to identify and get rid of tumor cells at the early stage of carcinogenesis, which results in tumor development. The mechanisms by which intestinal microbial metabolites, including short-chain fatty acids (SCFAs), microbial tryptophan catabolites (MTCs), polyamines (PAs), hydrogen sulfide, and secondary bile acids, are involved in tumorigenesis and development by regulating immune responses are summarized in this review. SCFAs and MTCs can prevent cancer by altering the expression of enzymes and epigenetic modifications in both immune cells and intestinal epithelial cells. MTCs can also stimulate immune cell receptors to inhibit the growth and metastasis of the host cancer. SCFAs, MTCs, bacterial hydrogen sulfide and secondary bile acids can control mucosal immunity to influence the occurrence and growth of tumors. Additionally, SCFAs, MTCs, PAs and bacterial hydrogen sulfide can also affect the anti-tumor immune response in tumor therapy by regulating the function of immune cells. Microbial metabolites have a good application prospect in the clinical diagnosis and treatment of tumors, and our review provides a good basis for related research.

Advancing Colorectal Cancer Diagnosis with AI-Powered Breathomics: Navigating Challenges and Future Directions.

Early detection of colorectal cancer is crucial for improving outcomes and reducing mortality. While there is strong evidence of effectiveness, currently adopted screening methods present several shortcomings which negatively impact the detection of early stage carcinogenesis, including low uptake due to patient discomfort. As a result, developing novel, non-invasive alternatives is an important research priority. Recent advancements in the field of breathomics, the study of breath composition and analysis, have paved the way for new avenues for non-invasive cancer detection and effective monitoring. Harnessing the utility of Volatile Organic Compounds in exhaled breath, breathomics has the potential to disrupt colorectal cancer screening practices. Our goal is to outline key research efforts in this area focusing on machine learning methods used for the analysis of breathomics data, highlight challenges involved in artificial intelligence application in this context, and suggest possible future directions which are currently considered within the framework of the European project ONCOSCREEN.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Targeting of tumor cells by custom antigen transfer: a novel approach for immunotherapy of cancer.

In the early stages of carcinogenesis, the transformed cells become "invisible" to the immune system. From this moment on, the evolution of the tumor depends essentially on the genotype of the primitive cancer cells and their subsequent genetic drift. The role of the immune system in blocking tumor progression from the earliest stages is largely underestimated because by the time tumors are clinically detectable, the immune system has already completely failed its task. Therefore, a clinical treatment capable of restoring the natural anti-tumor role of the immune system could prove to be the "ultimate weapon" against cancer. Herein, we propose a novel therapeutic approach for the treatment of solid cancer that exploits the capability of activated monocytes to transfer major histocompatibility complex I (MHC-I) molecules bound to antigenic peptides to cancer cells using microvesicles as cargo, making tumor cells target of a "natural" CD8+ T lymphocyte cytotoxic response.

Gut virome profiling identifies an association between temperate phages and colorectal cancer promoted by Helicobacter pylori infection

ABSTRACT Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. While a close correlation between chronic Helicobacter pylori infection and CRC has been reported, the role of the virome has been overlooked. Here, we infected Apc-mutant mouse models and C57BL/6 mice with H. pylori and conducted a comprehensive metagenomics analysis of H. pylori-induced changes in lower gastrointestinal tract bacterial and viral communities. We observed an expansion of temperate phages in H. pylori infected Apc +/1638N mice at the early stage of carcinogenesis. Some of the temperate phages were predicted to infect bacteria associated with CRC, including Enterococcus faecalis. We also observed a high prevalence of virulent genes, such as flgJ, cwlJ, and sleB, encoded by temperate phages. In addition, we identified phages associated with pre-onset and onset of H. pylori-promoted carcinogenesis. Through co-occurrence network analysis, we found strong associations between the viral and bacterial communities in infected mice before the onset of carcinogenesis. These findings suggest that the expansion of temperate phages, possibly caused by prophage induction triggered by H. pylori infection, may have contributed to the development of CRC in mice by interacting with the bacterial community.

Α Humanized RANKL Transgenic Mouse Model of Progestin-Induced Mammary Carcinogenesis for Evaluation of Novel Therapeutics.

Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.

Pellino 3 promotes the colitis-associated colorectal cancer through suppression of IRF4-mediated negative regulation of TLR4 signalling.

The incidence of colitis-associated colorectal cancer (CAC) has increased due to a high-nutrient diet, increased environmental stimuli and inherited gene mutations. To adequately treat CAC, drugs should be developed by identifying novel therapeutic targets. E3 ubiquitin-protein ligase pellino homolog 3 (pellino 3; Peli3) is a RING-type E3 ubiquitin ligase involved in inflammatory signalling; however, its role in the development and progression of CAC has not been elucidated. In this study, we studied Peli3-deficient mice in an azoxymethane/dextran sulphate sodium-induced CAC model. We observed that Peli3 promotes colorectal carcinogenesis with increased tumour burden and oncogenic signalling pathways. Ablation of Peli3 reduced inflammatory signalling activation at the early stage of carcinogenesis. Mechanistic studies indicate that Peli3 enhances toll-like receptor 4 (TLR4)-mediated inflammation through ubiquitination-dependent degradation of interferon regulatory factor 4, a negative regulator of TLR4 in macrophages. Our study suggests an important molecular link between Peli3 and colonic inflammation-mediated carcinogenesis. Furthermore, Peli3 can be a therapeutic target in the prevention and treatment of CAC.

230 Mutational landscape of normal human skin: Clues to understanding early-stage carcinogenesis in keratinocyte neoplasia

230 Mutational landscape of normal human skin: Clues to understanding early-stage carcinogenesis in keratinocyte neoplasia

Abstract 5751: Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis

Abstract Background The major genetic driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation in mouse models. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). Consequently, KrasG12D;Pdx1f/f (Pdx1 knockout) mice present with massively accelerated tumor formation. How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods To elucidate mechanisms responsible for the cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq from pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Expression of differentially regulated genes was validated by RNAscope and immunohistochemistry staining. The role of identified target genes was studied in pancreatic cancer cell lines. Results Single-cell ATAC-seq proved as a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. While sole expression of oncogenic Kras lead to reduced accessibility of acinar differentiation genes in acinar cells, these changes were much more prominent in KrasG12D;Pdx1f/f mice, promoting metaplastic conversion. Notably, acinar cells of KrasG12D;Pdx1f/f animals as well as a proportion of metaplastic lesions in both, KrasG12D and KrasG12D;Pdx1f/f mice, showed elevated accessibility and expression of the Ror2 gene. As a receptor protein tyrosine kinase, Ror2 controls essential signaling pathways, such as Ras-MAPK signaling. By analyzing Ror2 knockout mice, we found that the receptor kinase regulates the identity of metaplastic epithelia. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed ROR2 expression in a subset of cancer cells. Knockdown of ROR2 in pancreatic cancer cell lines significantly decreased cell proliferation, while overexpression induced a profound increase in proliferation and in epithelial-to-mesenchymal transition based on the downregulation of multiple epithelial markers and an upregulation of an array of mesenchymal genes. Conclusions Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 leads to vast alterations of acinar cell identity and significantly accelerated transformation. We identified induced expression of the receptor kinase Ror2, which regulates pancreatic cancer initiation and drives pancreatic cancer cell aggressiveness. Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, Ivonne Regel, Filip Bednar, Howard Crawford. Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5751.

Exercise through Shaking Stimuli Suppresses Cancer Growth via the Wnt pathway in ApcMin/+ Mice.

Exercise has been reported to suppress colorectal cancer; however, the mechanism of suppression by exercise and its effect on the Wnt pathway, which is particularly involved in the early stage of carcinogenesis, remain unclear. In this study, we subjected ApcMin/+ mice to exercise by shaking stimuli to investigate the mechanisms of suppressing colorectal cancer, and focused on the Ca2+ pathway, which is one of the β-catenin-independent Wnt signaling pathways that suppress the accumulation of β-catenin. Mice in the exercise group were subjected to exercise by shaking stimuli for 30 min/session, 6 sessions/ week, for a total of 11 weeks. The number and diameter of intestinal polyps were calculated. Expression analysis of β-catenin and Pak1 from the intestinal tract and Wnt5a-Pan and Wnt5a-Long from the gastrocnemius muscle was performed by western blotting. The expression of β-catenin and Wnt5a-Pan was observed by immunohistochemical staining. The levels of expression of β-catenin and Pak1 in the small intestine were low in the exercise group, indicating that exercise suppressed the accumulation of β-catenin. In the gastrocnemius muscle, the levels of expression of Wnt5a-Pan and Wnt5a-Long were significantly higher in the exercise group (p < 0.05). Histological analysis revealed that the percentage of large polyps was significantly lower in the exercise group than in the control group (p < 0.01), revealing that exercise suppressed the growth of polyps. In addition, the villi/crypt ratio (V/C ratio) was significantly higher in the exercise group, suggesting the suppression of exercise-induced local inflammation in the small intestine. We believe that the mechanism of polyp growth suppression is related to the inflammatory and not the Wnt pathway. This study clarified the growth-suppressing effect of a novel exercise method on cancer. We believe that its development and clinical application might open new possibilities for the prevention treatment of colorectal cancer.

Mutational Landscape of Normal Human Skin: Clues to Understanding Early-Stage Carcinogenesis in Keratinocyte Neoplasia

Normal skin contains numerous clones carrying cancer driver mutations. However, the mutational landscape of normal skin and its clonal relationship with skin cancer requires further elucidation. The aim of our study was to investigate the mutational landscape of normal human skin. We performed whole-exome sequencing using physiologically normal skin tissues and the matched peripheral blood (n= 39) and adjacent-matched skin cancers from a subset of patients (n= 10). Exposed skin harbored a median of 530 mutations (10.4/mb, range= 51-2,947), whereas nonexposed skin majorly exhibited significantly fewer mutations (median= 13, 0.25/mb, range= 1-166). Patient age was significantly correlated with the mutational burden. Mutations in six driver genes (NOTCH1, FAT1, TP53, PPM1D, KMT2D, and ASXL1) were identified. De novo mutational signature analysis identified a single signature with components of UV- and aging-related signatures. Normal skin harbored only three instances of copy-neutral loss of heterozygosity in 9q (n= 2) and 6q (n= 1). The mutational burden of normal skin was not correlated with that of matched skin cancers, and no protein-coding mutations were shared. In conclusion, we revealed the mutational landscape of normal skin, highlighting the role of driver genes in the malignant progression of normal skin.

PCDH17 gene promoter methylation status in a cohort of Egyptian women with epithelial ovarian cancer

Background and objectiveOvarian cancer is a leading cause of female mortality. Epigenetic changes occur in early stages of carcinogenesis and represent a marker for cancer diagnosis. Protocadherin 17 (PCDH17) is a tumor suppressor gene involved in cell adhesion and apoptosis. The methylation of PCDH17 gene promoter has been described in several cancers including ovarian cancer. The aim of the study was to compare the methylation status of PCDH17 gene promoter between females diagnosed with epithelial ovarian cancer and a control group composed of normal and benign ovarian lesions.MethodsFifty female subjects were included in our study (25 ovarian cancer patients and 25 controls). DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of the subjects. Methylation levels for six CpG sites in the PCDH17 gene promoter were assessed by pyrosequencing.ResultsThe methylation levels at five out of six sites were significantly higher in females with epithelial ovarian cancer compared to the control group. Moreover, the same applies for the mean methylation level with p value 0.018.ConclusionMethylation of PCDH17 gene promoter plays a role in ovarian carcinogenesis and can be used for diagnosis and early detection.

Novel Non-Cyclooxygenase Inhibitory Derivative of Sulindac Inhibits Breast Cancer Cell Growth In Vitro and Reduces Mammary Tumorigenesis in Rats.

The nonsteroidal anti-inflammatory drug (NSAID) sulindac demonstrates attractive anticancer activity, but the toxicity resulting from cyclooxygenase (COX) inhibition and the suppression of physiologically important prostaglandins precludes its long-term, high dose use in the clinic for cancer prevention or treatment. While inflammation is a known tumorigenic driver, evidence suggests that sulindac's antineoplastic activity is partially or fully independent of its COX inhibitory activity. One COX-independent target proposed for sulindac is cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) isozymes. Sulindac metabolites, i.e., sulfide and sulfone, inhibit cGMP PDE enzymatic activity at concentrations comparable with those associated with cancer cell growth inhibitory activity. Additionally, the cGMP PDE isozymes PDE5 and PDE10 are overexpressed during the early stages of carcinogenesis and appear essential for cancer cell proliferation and survival based on gene silencing experiments. Here, we describe a novel amide derivative of sulindac, sulindac sulfide amide (SSA), which was rationally designed to eliminate COX-inhibitory activity while enhancing cGMP PDE inhibitory activity. SSA was 68-fold and 10-fold less potent than sulindac sulfide (SS) in inhibiting COX-1 and COX-2, respectively, but 10-fold more potent in inhibiting growth and inducing apoptosis in breast cancer cells. The pro-apoptotic activity of SSA was associated with inhibition of cGMP PDE activity, elevation of intracellular cGMP levels, and activation of cGMP-dependent protein kinase (PKG) signaling, as well as the inhibition of β-catenin/Tcf transcriptional activity. SSA displayed promising in vivo anticancer activity, resulting in a 57% reduction in the incidence and a 62% reduction in the multiplicity of tumors in the N-methyl-N-nitrosourea (MNU)-induced model of breast carcinogenesis. These findings provide strong evidence for cGMP/PKG signaling as a target for breast cancer prevention or treatment and the COX-independent anticancer properties of sulindac. Furthermore, this study validates the approach of optimizing off-target effects by reducing the COX-inhibitory activity of sulindac for future targeted drug discovery efforts to enhance both safety and efficacy.