Adenoma Formation Research Articles

Shen-Bai-Jie-Du decoction suppresses the progression of colorectal adenoma to carcinoma through regulating gut microbiota and short-chain fatty acids

BackgroundShen-Bai-Jie-Du decoction (SBJDD), a traditional Chinese herb formula developed based on evidence-based medicine, is efficacy to reduce the recurrence and carcinogenesis of colorectal adenoma. However, the mechanism of SBJDD to treat colorectal adenoma remains unclear. The present study aims to investigate the efficacy and mechanism of SBJDD on colorectal adenoma carcinogenesis from the aspects of regulating gut microbiota and short-chain fatty acids (SCFAs).MethodsTwenty-one patients diagnosed with colorectal adenoma were recruited in the study and required to take SBJDD for four consecutive weeks. Analysis of gut microbiota was conducted using 16S rRNA gene amplicon sequencing, while levels of SCFAs in fecal and serum samples were determined through HPLC–MS/MS. Additionally, twenty-four Apcmin/+ mice were randomly assigned to normal diet (ND), high-fat diet (HFD), and SBJDD groups. The pharmacological effects and mechanism of SBJDD on colorectal adenoma carcinogenesis were assessed using RT-qPCR, HE staining, IHC staining, Western blot, IF staining, and Flow cytometry assays.ResultsOur clinical study has shown that SBJDD can regulate the gut microbiota composition and enhance SCFAs production in patients with colorectal adenoma. SBJDD alleviated colorectal adenoma formation and carcinogenesis, as well as protected the integrity of the intestinal barrier in the Apcmin/+ mice model compared to the HFD group. Additionally, SBJDD was found to regulate gut microbiota capable of producing SCFAs. G protein-coupled receptors GPR43, GPR41, and GPR109a were effectively activated in the SBJDD group, while HDAC1 and HDAC3 were inhibited. Furthermore, decreased expression levels of interleukin 1 beta (IL-1β) and interleukin 6 (IL-6), along with elevated expression level of interleukin 10 (IL-10), were observed in the colorectal tissue of the SBJDD group. Finally, SBJDD exhibited the ability to reduce the proportion of M1-type macrophages while increasing the proportion of M2-type macrophages.ConclusionsOur study objectively demonstrated the pharmacological effects of SBJDD in inhibiting the progression of colorectal adenoma and investigated its mechanisms in terms of regulating gut microbiota, increasing SCFAs, and reducing colorectal inflammation.

6530 A Case of Lymphocytic Hypophysitis Followed by Acromegaly

Abstract Disclosure: L. Medina Mora: None. S. Htoo: None. A. Sanchez Ruiz: None. M. Harshan: None. M. Liu: None. Introduction: Acromegaly, a disorder with an estimated incidence of 0.28-0.4 cases per 100,000 individuals, is characterized by overproduction of growth hormone. Features include acral enlargement, frontal bossing, macrognathia, headaches, and hyperhidrosis. Comorbidities include hypertension, sleep apnea, type 2 diabetes, and cardiomyopathies. Hypophysitis, with an incidence of 1 in 9 million individuals, is an even rarer condition that involves pituitary gland inflammation. It can often lead to hormonal dysfunction and compression of surrounding structures. It is extremely rare for both acromegaly and hypophysitis to occur in the same individual. In this case report, we present a patient who developed lymphocytic hypophysitis followed by acromegaly. Case Report: A 58-year-old woman with a history of hypertension and lymphocytic hypophysitis (discovered during a workup of headaches without hormone derangement) returned to the clinic two years after complaining of progressive enlargement of her hands and feet, fragmented sleep, insomnia, and coarsening of facial features. She was found to have elevated IGF-1 (886 ng/mL; reference range: 48-235 ng/mL) and growth hormone levels (86.9 ng/mL; reference range: 0.12-9.88 ng/mL). An MRI showed a 2.9 x 3.2 x 3.8 cm suprasellar/sellar mass causing pressure on the optic chiasm, hypothalamus, third ventricle and midbrain. The patient underwent transsphenoidal resection (TSR), which was incomplete because the tumor was located behind the pituitary gland, followed by a right craniotomy to remove the remaining tumor. The tumor specimen stained positive for GH and PIT 1, exhibiting elevated proliferative indices (>2 mitotic figures per 10HPF, Ki-67 20%) and p53 labeling (5%). After the surgery, the patient's IGF-1 and GH levels normalized. However, her postoperative course was complicated by third cranial nerve palsy, diabetes insipidus, hypothyroidism, and secondary adrenal insufficiency. Discussion: Although TSR is the first-line treatment for most patients with acromegaly, it may not always result in a complete cure, as almost 50% of patients with macroadenomas do not achieve full surgical recovery. For persistent disease, medical therapy or transcranial surgery may be required. The tumor in this case was atypically proliferative, which was likely why GH and IGF-1 levels rose rapidly, manifesting in acromegalic symptoms in our patient two years after diagnosis of lymphocytic hypophysitis. The natural history of hypophysitis typically progresses from inflammation to fibrosis and subsequently to atrophy of the pituitary cells. This atrophy may cause changes in regulatory hormones that promote tumorigenesis. Therefore, hypophysitis may stimulate adenoma formation leading to acromegaly. More research is needed to characterize the relationship between these two rare entities. Presentation: 6/2/2024

Identifying targetable metabolic dependencies across colorectal cancer progression

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

E-cigarettes increase the risk of adenoma formation in murine colorectal cancer model.

E-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development. A mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APCMin/+, leads to the development of colon adenomas within 16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2-hour exposures per day (1 hour of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA. CPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers (CD44, Lgr-5, DCLK1, and Ki67). The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone. E.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects.

The Interplay among Wnt/β-catenin Family Members in Colorectal Adenomas and Surrounding Tissues.

The colorectal adenoma undergoes neoplastic progression via the normal epithelium-adenoma-adenocarcinoma sequence as reported in the Vogelgram. The hazard of developing a tumor is deeply associated with the number and size of adenomas and their subtype. Adenomatous polyps are histologically categorized as follows: approximately 80-90% are tubular, 5-15% are villous, and 5-10% are tubular/villous. Given the higher risk of a malignant transformation observed in tubular/villous adenomas, patients diagnosed with adenomatous polyposis are at an improved risk of developing CRC. The Wnt/β-catenin pathway plays a key role in the onset of colorectal adenoma; in particular, intestinal cells first acquire loss-of-function mutations in the APC gene that induce the formation of adenomas. Wnt/β-catenin pathway APC, Wnt3a, Wnt5a, LEF1, and BCL9 genes and protein expression analyses were conducted by qRT-PCR and western blot in 68 colonic samples (polyps and adjacent mucosa) from 41 patients, of which 17 were affected by FAP. Ten normal colonic mucosal samples were collected from 10 healthy donors. In this study, both the APC gene and protein were less expressed in the colon tumor compared to the adjacent colonic mucosa. Conversely, the activated β-catenin was more expressed in polyps than in the adjacent mucosa. All results confirmed the literature data on carcinomas. A statistically significant correlation between Wnt3a and BCL9 both in polyps and in the adjacent mucosa underlines that the canonical Wnt pathway is activated in early colon carcinogenesis and that the adjacent mucosa is already altered. This is the first study analyzing the difference in expression of the Wnt/β-catenin pathway in human colorectal adenomas. Understanding the progression from adenomas to colorectal carcinomas is essential for the development of new therapeutic strategies and improving clinical outcomes with the use of APC and β-catenin as biomarkers.

The two faces of Blastocystis spp.: is it the cause of colorectal cancer (CRC) or a consequence of it?

IntroductionOver the last few years, there has been an increase in the prevalence of <i>Blastocystis</i> spp. in colorectal cancer (CRC) patients. Moreover, various in vitro and in vivo studies have highlighted that intestinal colonisation of <i>Blastocystis</i> spp. has an influence on host immune responses leading to cellular apoptosis and membrane permeability. It has been suggested that <i>Blastocystis</i> spp. is an important risk factor for the worsening of CRC.AimTo present evidence concerning the association between CRC and <i>Blastocystis</i> spp.Material and methodsA review of the literature was performed by searching Science Direct, PubMed, Scopus and Google Scholar databases up to December, 2023.Results and discussionOut of all in vitro and in vivo studies selected for this review, the majority of them have confirmed a significantly higher prevalence of <i>Blastocystis</i> spp. in colorectal cancer patients in comparison to the control groups. Several in vitro human colorectal carcinoma cell line studies have shown significant cytopathic and immunological effects of <i>Blastocystis</i> spp. Additionally, in vivo experimental animal model studies have shown that <i>Blastocystis</i> spp. infection significantly contributed to large intestinal polyp (colorectal adenoma) formation and the progression of colorectal carcinogenesis.ConclusionsThese studies strongly support suggestions that <i>Blastocystis</i> spp. could be an important factor to existing CRC development by influencing the host immune response and increasing oxidative damage.

Clinical aspects of the plurihormonal pituitary adenoma formation

Generally pituitary tumors have a benign growth, however, there are difficulties in diagnosis and treatment because of non-specific symptoms and the inability to predict the tumor growth. In clinical practice a hormonal activity of tumors has the significant role. To a greater extent, pituitary adenomas are prolactinomas, but the hypersecretion of prolactin could be combined with an excessive production of somatotropic hormone. In this case, the clinical picture of hyperprolactinemia is accompanied by acromegaly symptoms. The presented clinical case demonstrates the main reasons for the clinical appointment such as menstrual cycle disorders, prenatal preparation. A hyperprolactinemia has been detected, but as a treatment result, drug compensation was achieved and pregnancy occurred, then the woman gave birth without obstetric complications. Subsequently, other complaints arose, which expanded the diagnostic search and revealed the presence of a plurihormonal tumor.

Effect of vitamin E δ-tocotrienol and aspirin on Wnt signaling in human colon cancer stem cells and in adenoma development in APCmin/+ mice.

In this study, we evaluated the effects of vitamin E δ-tocotrienol (DT3) and aspirin on Wnt signaling in human colon cancer stem cells (CCSCs) and in the prevention of adenoma formation in APCmin/+ mice. We found that knockdown of the adenomatous polyposis coli (APC) gene led to subsequent activation of Wnt signaling in colon epithelial cells (NCM460-APCsiRNA) and induction of β-catenin and its downstream target proteins c-MYC, cyclin D1, and survivin. When aspirin and DT3 were combined, cell growth and survival were inhibited and apoptosis was induced in colon epithelial cells and in CCSCs. However, DT3 and/or aspirin had little or no effect on control normal colon epithelial cells (NCM460-NCsiRNA). The induction of apoptosis was directly related to activation of caspase 8 and cleavage of BID to truncated BID. In addition, DT3 and/or aspirin-induced apoptosis was associated with cleaved PARP, elevated levels of cytosolic cytochrome c and BAX, and depletion of anti-apoptotic protein BCl-2 in CCSCs. The combination of aspirin and DT3 inhibited the self-renewal capacity, Wnt/β-catenin receptor activity, and expression of β-catenin and its downstream targets c-MYC, cyclin D1 and survivin in CCSCs. We also found that treatment with DT3 alone or combined with aspirin significantly inhibited intestinal adenoma formation and Wnt/ β-catenin signaling and induced apoptosis, compared to vehicle, in APCmin/+ mice. Our study demonstrated a rationale for further investigation of the combination of DT3 and aspirin for colorectal cancer prevention and therapy.

Morphological Changes and Inflammation Preceded the Pathogenesis of 1,2-Dimethylhydrazine-Induced Colorectal Cancer.

This study examined the morphological changes in the colonic mucosa and the presence of inflammation in rats induced with 1,2-dimethylhydrazine (DMH) 30 mg/kg BW over 9, 11, and 13 weeks without a latency period. Hematoxylin and eosin staining was performed to assess the morphology and characteristic alteration of the epitheliocytes in the colon. Immunohistochemistry was employed to assess the expression of tumor necrosis factor (TNF)-α and cyclooxygenase-2 (COX-2). The difference in the severity of inflammation and COX-2 expression was examined using one-way analysis of variance. The correlation of COX-2 expression with the severity of inflammation was analyzed using Spearman's rank correlation test. Until week 13, chronic inflammation and non-hyperplastic and hyperplastic aberrant crypt foci occurred. The severity of inflammation gradually shifted from high moderate to low moderate. TNF-α expression was high in all groups; however, COX-2 expression was gradually lower with longer duration of induction, which corresponded with the severity of inflammation. DMH induction until week 13 without a latency period caused chronic inflammation without the formation of adenoma or adenocarcinoma. A very strong correlation was established between COX-2 expression and inflammation.

The Role of Decreased Expression of Glucocorticoid Receptors in the Development of Itsenko-Cushing Syndrome

Background: Itsenko-Cushing syndrome is caused by unregulated excessive cortisol incretion by the adrenal cortex. The causes of increased stimulation of cortisol incretion by the adrenal cortex, leading to hyperplasia and tumors (adenoma and cancer) of the adrenal cortex, as well as to adenoma of the pituitary gland itself, remain unclear to date. Materials and Methods: 6 patients with adrenal Itsenko-Cushing syndrome with adrenal cortex adenoma were under observation. The age of the patients ranged from 24 to 87 years. The patients underwent removal of the adrenal gland on the affected side. The control group consisted of 6 individuals who underwent surgical treatment of varicocele and hernias of various localization. In patients of the main group and control group, with their consent, a small fragment of skin from the edge of the surgical wound was taken for examination, which were fixed in a 10% formalin solution. Results: The presence of adrenal Itsenko-Cushing syndrome in patients of the main group was confirmed by laboratory and instrumental studies, as well as characteristic clinical manifestations. The average Histochemical score of the expression of glucocorticoid receptors (GR) of the skin in patients of the main group with Itsenko-Cushing adrenal syndrome was 2.7 times less than the same value in the control group. Conclusions: A decrease in the formation or absence of glucocorticoid receptors (congenital or acquired) blocks the negative feedback of cortisol suppression of corticoliberin production by the hypothalamus and ACTH-pituitary gland. The resulting excessive incretion of corticoliberin becomes the main link in pathogenesis of pituitary adenoma formation, and an excessive incretion of ACTH leads to the formation of cortical hyperplasia and tumors (adenomas, cancers) of the adrenal cortex. Unregulated excessive cortisol production by hyperplastic adrenal tissues or benign/malignant adrenal tumors leads to overcoming resistance to glucocorticoids and, according to the principle of negative feedback, suppresses the production of corticoliberin by the hypothalamus and ACTH-pituitary gland. As a result, the pituitary form of Itsenko-Cushing syndrome turns into an adrenal form with an unregulated increase in cortisol levels and a decrease in ACTH levels. Transfusion of the mononuclear fraction of peripheral blood obtained from healthy donors, which allows the pluripotent stem cells contained in it to partially replace the altered cells of all tissues of patients, is a promising method of treating patients with Itsenko-Cushing syndrome due to a hereditary decrease in the expression of glucocorticoid receptors.

Interaction of Colorectal Neoplasm Risk Factors and Association with Metabolic Health Status Focusing on Normal Waist-to-Hip Ratio in Adults.

We aimed to evaluate the interaction between colorectal adenoma risks among asymptomatic individuals in terms of metabolic health status and obesity, and examine the normal waist-to-hip ratio (WHR) in adults with colorectal adenoma risk. A cross-sectional, retrospective study was conducted at MacKay Memorial Hospital involving 16,996 participants who underwent bidirectional gastrointestinal endoscopy between 2013 and 2023. The study recorded important clinicopathological characteristics, including age, body mass index and WHR, Framingham Risk Score (FRS), blood glucose level, and Helicobacter pylori (H. pylori) infection status. Multivariate logistic regression analysis demonstrated that elevated hemoglobin A1C (HbA1c), increased FRS, positive H. pylori infection, and WHR ≥ 0.9 are independent risk factors for colorectal adenoma. In examining the interaction between FRS and WHR using multivariate logistic regression to evaluate adenoma risk, the OR for the interaction term was 0.95, indicating a decline in adenoma risk when considering the interaction between these two factors. Incorporating HbA1c into the analysis, evaluating the interaction between FRS and WHR still demonstrated a statistically significant impact on adenoma risk (OR 0.96, p < 0.001). Participants with WHR < 0.9, elevated FRS, positive H. pylori infection, and increased HbA1c levels were associated with a higher risk of colorectal adenoma formation. Remarkably, the increased risk of adenoma due to rising HbA1c levels was statistically significant only for those with a WHR < 0.9. An increase in FRS and HbA1c or a positive H. pylori infection still warrants vigilance for colorectal adenoma risk when WHR is 0.9. These factors interacted with each other and were found to have a minimal decline in adenoma risk when considering the interaction between WHR and FRS.

Abstract SY07-02: Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut

Abstract The epithelial monolayer of the intestine is one of the fastest renewing tissues of the human body. Every week, the entire epithelium is replaced by intestinal stem cells (ISCs) that reside in crypt-like structures within the monolayer. Within these crypts, a small number of ISCs continuously divide to provide all specialized cell types necessary to maintain normal intestinal homeostasis, while simultaneously competing with each other for a position within the crypt bottom. This competition, characterized by neutral loss-and-replacement events, can be drastically disturbed when one of the ISCs acquires a mutation that confers a competitive advantage. As a result, mutant ISCs will replace all normal ISCs, colonize the intestinal crypt, and facilitate the initiation of colorectal cancer (CRC). CRC development is a stepwise process characterized by the accumulation of mutations in cancer driver genes over time. Almost 80% of all CRCs present with early mutations in the tumor suppressor gene APC, leading to constitutive activation of the Wnt pathway and the formation of pre-malignant adenomas. We have previously demonstrated that, within individual crypts, Apc-mutant ISCs have a competitive advantage over normal ISCs, although the mechanism underlying this unequal competition remained unclear. Recently, we revealed that Apc-mutant ISCs in fact act as "supercompetitors" by secreting Wnt antagonists such as NOTUM, WIF1 and DKK2, that actively disadvantage neighboring wild type ISCs by forcing them do differentiate. We demonstrated that boosting wild type fitness by downstream pharmacological activation of the Wnt signaling pathway by GSK3β-inhibitor lithium rendered wild type ISCs resistant to the Wnt antagonists, reduced the number of Apc-mutant crypts, and prevented adenoma formation. Together, this work highlights that modulating cell competition between normal and mutant cells within the crypt can provide a novel approach to prevent CRC development. Our findings are particularly relevant for familial adenomatous polyposis (FAP) patients, that carry germline mutations in APC and are predisposed to the development of CRC. Based on our fundamental discoveries, we are currently performing a clinical trial to assess the chemopreventive effect of lithium on the expansion of APC-mutant clones in FAP patients [ClinicalTrials.gov: NCT05402891]. Importantly, it is becoming increasingly evident that intestinal adenomas do not form from a single Apc-mutant crypt, but require fields of multiple mutant crypts to facilitate tumor initiation. Although field cancerization by enhanced crypt fission has been observed for several oncogenic mutants in the gut (e.g. KrasG12D, PIK3CAH1047R), Apc-mutant cells are often contained within individual crypts. This indicates that multiple independent mutant clones may act cooperatively to facilitate tumorigenesis, thereby pointing towards a polyclonal origin of intestinal adenomas. Our current work investigates how Apc-mutant clones interact by modifying their local environment through reciprocal signalling with stromal and epithelial cells to escape the anatomical constraints of the crypt, thereby unlocking their oncogenic potential in the gut. Ultimately, unravelling the molecular mechanisms underlying clonal cooperation will inform potential chemoprevention strategies for CRC. Citation Format: Sanne van Neerven. Cell competition vs cell cooperation: Unlocking oncogenic potential of mutant clones in the gut [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY07-02.

Telomere dysfunction alters intestinal stem cell dynamics to promote cancer

Telomere dynamics are linked to aging hallmarks, and age-associated telomere loss fuels the development of epithelial cancers. In Apc-mutant mice, the onset of DNA damage associated with telomere dysfunction has been shown to accelerate adenoma initiation via unknown mechanisms. Here, we observed that Apc-mutant mice engineered to experience telomere dysfunction show accelerated adenoma formation resulting from augmented cell competition and clonal expansion. Mechanistically, telomere dysfunction induces the repression of EZH2, resulting in the derepression of Wnt antagonists, which causes the differentiation of adjacent stem cells and a relative growth advantage to Apc-deficient telomere dysfunctional cells. Correspondingly, in this mouse model, GSK3β inhibition countered the actions of Wnt antagonists on intestinal stem cells, resulting in impaired adenoma formation of telomere dysfunctional Apc-mutant cells. Thus, telomere dysfunction contributes to cancer initiation through altered stem cell dynamics, identifying an interception strategy for human APC-mutant cancers with shortened telomeres.

Downregulation of ABCC3 activates MAPK signaling through accumulation of deoxycholic acid in colorectal cancer cells.

ABCC3 (also known as MRP3) is an ATP binding cassette transporter for bile acids, whose expression is downregulated in colorectal cancer through the Wnt/β-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. Gene expression analysis of colorectal adenoma isolated from familial adenomatous polyposis patients revealed that genes related to bile acid secretion including ABCC3 were downregulated as early as at the stage of adenoma formation. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid, a secondary bile acid, respectively, in colorectal cancer cells. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that nonsteroidal anti-inflammatory drugs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of familial adenomatous polyposis. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling.

Abstract 3938: A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas

Abstract Gain-of-function oncogenic mutations in RIT1 have been identified in lung adenocarcinoma and are mutually exclusive with other reported driver mutations, highlighting the role of RIT1 as a cancer driver. RIT1 is a poorly-studied non-classical RAS GTPase and, to date, there are no reported genetically-engineered mouse models of RIT1-driven lung adenocarcinoma. Here, we have generated a conditional knock-in allele within the endogenous mouse Rit1 locus, introducing the most frequent mutation observed in lung adenocarcinomas (RIT1M90I). Upon intranasal administration of adenovirus encoding the Cre recombinase to induce somatic recombination in the mouse lungs, we observed the formation of discrete adenomas. Although Rit1M90I mutation can promote lung tumor initiation in mice, we found that this phenotype is less penetrant than the common KrasG12D mutation. Immunohistochemistry analysis of Rit1M90I and KrasG12D tumors showed expression of typical histological markers associated with lung adenocarcinoma (i.e., TTF1 and SPC), as well as increased levels of Ki67 and pERK expression. Next, to develop potential pharmacological strategies for RIT1-driven lung tumors, we first explored drugs that inhibit the RAS/MAPK pathway and are either FDA-approved or under clinical development. In vitro experiments with cell lines carrying the RIT1M90I mutation demonstrated high sensitivity to SHP2, MEK, RAF, and RAS inhibitors. In vivo, SHP2 and RAS, but not MEK, inhibitors slowed down tumor progression in both cell line and patient-derived xenografts. Our results indicate that, although RAS signaling might be needed for RIT1-dependent growth, these tumors are not sensitive to MAPK inhibition as a monotherapy and, hence, other pathways could contribute to tumor growth. Therefore, we decided to explore whether targeting RIT1 directly, could be a more beneficial strategy for these tumors. In order to inhibit oncogenic RIT1 directly, we have undertaken two complementary approaches. First, because RIT1 mutations were shown to impair its proteolysis, we explored whether restoring RIT1 proteasomal degradation could be a promising strategy. Recently, the development of Proteolysis Targeting Chimeras (PROTAC) has demonstrated that targeted degradation can be used as a pharmacological approach for the treatment of cancer. Therefore, we have tested successfully several proof-of-concept PROTAC approaches that caused degradation of oncogenic RIT1 in different cellular models. Second, we used a chemical biology approach to assess whether covalent inhibitors directed against the closely-related GTPase KRAS could cross-react and bind to oncogenic RIT1. We have identified a molecule that binds RIT1 and we are currently characterizing it in our preclinical models. Overall, our work provides comprehensive insights into the oncogenic role of RIT1 in lung cancer emphasizing its potential as a therapeutic target. Citation Format: Alessandro Mauro Mozzarelli, Antonio Cuevas-Navarro, Anatoly Urisman, Pau Castel. A novel mouse model reveals potential therapeutic strategies for RIT1-driven lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3938.

Abstract 1061: Diagnostic application of a novel blood CDH17 immunoassay in the detection of colorectal adenoma

Abstract Background: Colorectal cancer (CRC) is the most prevalent gastrointestinal (GI) cancer and contribute significantly to global cancer deaths. The mechanism of CRC development is a complex multistage process from hyperplastic lesions to the formation of adenomas, followed by the progression into malignancy. Hence, early detection during hyperplastic and adenomatous stages warrants new screening tests for effective interception of malignancy progression. Cadherin-17 (CDH17) is a biomarker characterized by its overexpression in several GI cancers, but restricted expression in intestinal mucosal epithelium of normal tissues. We have developed a blood immunoassay using a panel of RUO grade antibodies that unveiled CDH17 as a potential marker with high detection sensitivity and specificity for detecting adenomas. Our immunoassay is useful in identifying high-risk individuals with adenomas that may develop into CRC, which can significantly improve the diagnostic efficiency of colonoscopy that is currently lacking. Methods: The study recruited 66 patients (54 adenoma and 12 hyperplasia) and 39 normal samples (no abnormality by colonoscopy). CDH17 level in plasma samples were measured using the CDH17 immunoassay. Results: The novel CDH17 diagnostic assay showed an operating dynamic range from 90ng to 123pg. We observed a progressive and significant increase in the circulating CDH17 level from normal samples to the hyperplasia group, followed by the adenoma group (mean: 1.66ng/mL, 3.20ng/mL, 4.93ng/mL, respectively P&amp;lt;0.0001). The assay performance by ROC curve demonstrated an AUC of &amp;gt;0.9, with a sensitivity of 85% and specificity of 86%. NPV and PPV were 84% and 87%, respectively. The assay outperforms two market available FIT-DNA tests at predicting adenomas (Table. 1). Conclusion: The novel CDH17 immunoassay is potentially a powerful tool for the detection of aberrant plasma CDH17 expression, improving the surveillance and the detection of high-risk individuals for CRC. CDH17 immunoassay outperforms market availalble FIT-DNA tests at detecting hyperplasia and adenoma FIT-DNA test 1 FIT-DNA test 2 CDH17 blood immunoassay Age 40-74 Age 50-84 Age 45-93 Sensitivity (%) Advanced adenoma 63.5 42.4 85.0 Non-advanced adenoma (i.e Hyperplasia) Unavailable 17.2 58.3 Specificity (%) Normal control (no abnormality by colonoscopy) 87.1 89.8 86.1 NPV (%) Advanced adenoma Unavailable Unavailable 83.8 Non-advanced adenoma (i.e Hyperplasia) Unavailable Unavailable 86.1 PPV (%) Advanced adenoma Unavailable Unavailable 87.2 Non-advanced adenoma (i.e Hyperplasia) Unavailable Unavailable 58.3 Citation Format: Ng Lui, Felix Hon, Stella Sun, Pauline Leung, David Ho, John Moon Luk, Dominic Chi-Chung Foo. Diagnostic application of a novel blood CDH17 immunoassay in the detection of colorectal adenoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1061.

Abstract 1479: ABCC3 (MRP3)-mediated export of deoxycholic acid regulates MAPK signaling in colorectal adenoma and adenocarcinoma

Abstract ABCC3 (also known as MRP3) is an ATP binding cassette transporter and it works as a bile acids transporter. We previously elucidated that ABCC3 expression is downregulated in colorectal cancer through the Wnt/β-catenin signaling pathway. However, it remained unclear how downregulation of ABCC3 expression contributes to colorectal carcinogenesis. We here explored the role of ABCC3 in the progression of colorectal cancer-in particular, focusing on the regulation of bile acid export. First, we performed experiments with clinical samples of familial adenomatous polyposis (FAP) patients to confirm whether ABCC3 expression is already downregulated in colorectal adenomas. Next, we investigated the effect of ABCC3 expression on intracellular bile acid concentrations using ABCC3 knockdown and overexpression colorectal cancer cell lines (HT-29, SW620). The intracellular concentration of deoxycholic acid, which is known to have carcinogenic effects, was verified with fluorescently labeled deoxycholic acid. Then, we tested whether activation of oncogenic signaling by deoxycholic acid is affected by ABCC3 expression changes. Finally, we examined the effect of nonsteroidal anti-inflammatory drugs (NSAIDs), which have been suggested to inhibit the carcinogenesis of FAP, on the expression of ABCC3 in experiments with colorectal cancer cell lines. Gene and protein expression analysis of colorectal adenomas isolated from FAP patients revealed that ABCC3 were downregulated as early as at the stage of adenoma formation, and many other genes related to bile acid efflux were also downregulated. Knockdown or overexpression of ABCC3 increased or decreased intracellular concentration of deoxycholic acid in colorectal cancer cells, respectively. Forced expression of ABCC3 suppressed deoxycholic acid-induced activation of MAPK signaling. Finally, we found that several NSAIDs increased ABCC3 expression in colorectal cancer cells, suggesting that ABCC3 could be one of the targets for therapeutic intervention of FAP. Our data thus suggest that downregulation of ABCC3 expression contributes to colorectal carcinogenesis through the regulation of intracellular accumulation of bile acids and activity of MAPK signaling. Citation Format: Yukihiro Sato, Minoru Kobayashi, Masahiro Ohira, Ryo Funayama, Masamitsu Maekawa, Hideaki Karasawa, Shinobu Ohnuma, Michiaki Unno, Keiko Nakayama. ABCC3 (MRP3)-mediated export of deoxycholic acid regulates MAPK signaling in colorectal adenoma and adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1479.

Abstract 4780: TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death inthe United States and screening is the most effective method to reduce mortality. Chemoprevention, which aims to prevent CRC through eradication or prevention of precursor colonic adenomas, represents an alternative strategy to address this problem. The imipridone dordaviprone (ONC201) has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201 would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC201 in two adenoma-derived organoid lines: one developed from an adenoma obtained from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC201 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC201 (concentration ranging from 0 uM to 20 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from organoids treated with either 1.69 uM DMSO, 1.69 uM ONC201, or 3.38 uM ONC201 for FAP and 1.23 uM DMSO, 1.23 uM ONC201, or 2.46 uM ONC201 for SSA for 0, 24, or 48 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either 845 nM DMSO, 845 nM ONC201, or 1.69 uM ONC201 for FAP and 615 nM DMSO, 615 nM ONC201, or 1.23 uM ONC201 for SSA. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72hours. Results: ONC201 demonstrated antineoplastic effects in adenoma-derived FAP and SSA organoids. Half maximal inhibitory concentrations (IC50) of ONC201 in FAP and SSA organoids were 1.69 uM and 1.23 uM, respectively. As was noted in rodent studies, the mechanism of action of ONC201 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 were both upregulated) and the integrated stress response (ATF4 was upregulated). Co-culture with NK cells revealed an increase in NK-mediated organoid cell death. The ability of ONC201 to enhance NK-mediated killing is still being evaluated. Further analysis of the effects of ONC201 on stemness markers and on T-cell-mediated organoid cell killing are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC201. Citation Format: Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4780.

Early results of the integrative epigenomic-transcriptomic landscape of colorectal adenoma and cancer.

Aberrant methylation is common during the initiation and progression of colorectal cancer (CRC), and detecting these changes that occur during early adenoma (ADE) formation and CRC progression has clinical value. To identify potential DNA methylation markers specific to ADE and CRC. Here, we performed SeqCap targeted bisulfite sequencing and RNA-seq analysis of colorectal ADE and CRC samples to profile the epigenomic-transcriptomic landscape. Comparing 22 CRC and 25 ADE samples, global methylation was higher in the former, but both showed similar methylation patterns regarding differentially methylated gene positions, chromatin signatures, and repeated elements. High-grade CRC tended to exhibit elevated methylation levels in gene promoter regions compared to those in low-grade CRC. Combined with RNA-seq gene expression data, we identified 14 methylation-regulated differentially expressed genes, of which only AGTR1 and NECAB1 methylation had prognostic significance. Our results suggest that genome-wide alterations in DNA methylation occur during the early stages of CRC and demonstrate the methylation signatures associated with colorectal ADEs and CRC, suggesting prognostic biomarkers for CRC.

Molecular mechanisms of colorectal cancer formation and their significance (literature review)

Colorectal cancer remains an urgent problem of oncological proctology and requires active efforts to study risk factors, mechanisms for the formulation and search for genetic components that play a key role in determining the prognosis of the disease. This article analyzes the molecular mechanisms of colorectal cancer formation, factors of transformation of adenoma formation, pathogenesis and molecular nature of CRC. Subtypes based on transcription signatures allow for better refinement and provide insight into the development of subtype-specific treatment methods, which, in turn, can contribute to more effective treatment of this disease.