Wnt-beta Catenin Signaling Research Articles

DIPG-86. INVESTIGATING THE TUMOR ENVIRONMENT AND SIGNALING DEPENDENCIES DRIVING DIFFUSE MIDLINE GLIOMA TUMOR INITIATION, PROGRESSION, AND RESISTANCE: A COMPREHENSIVE APPROACH LEVERAGING BULK MULTIOMICS DECONVOLUTION AND SINGLE-CELL OMICS VALIDATION

Abstract BACKGROUND Diffuse Midline Glioma (DMG) is a incurable tumor affecting children. Recent genomic investigations have identified a recurrent H3K27M mutation which induces global alterations in histone methylation patterns and DNA methylation. These epigenetic modifications hint at pivotal roles in DMG pathogenesis, yet effective therapeutic strategies remain elusive, with median survival rates stagnant at approximately one year. This shortfall stems from two main factors: 1) Inadequate multiomics studies hindering our understanding of DMG evolution and tumor progression, and 2) Limited comprehension of the tumor microenvironment in DMG. METHODS To elucidate DMG’s clonal evolution, we adopted a comprehensive multi-region sampling approach, acquiring 33 specimens from seven patients. Whole-exome and transcriptome sequencing, alongside DNA methylation profiling, were performed. Additionally, we generated one of the most extensive single-cell multiomics datasets (>300,000 cells from 21 additional samples), with a focus on pre- and post-treatment, and dissemination effects in DMG. RESULTS Our analysis delineated a tumor-promoting microenvironment characterized by hypoxia and pro-inflammatory conditions, nurturing genomic alterations and specialized biological processes such as proliferation and epithelial-mesenchymal transition. Notably, a majority of immune cells exhibited M1-like polarization, bolstering pro-inflammatory programs within the tumor milieu. Subclones within the DMG cohort demonstrated dissemination potential, often exhibiting up-regulation of NOTCH, P53, and WNT beta-catenin signaling pathways. These findings suggest that DMG clones harboring dissemination capabilities outside the pons acquire additional phenotypic features, possibly mediated by epigenetic or transcriptional alterations, contributing to enhanced migratory and aggressive behaviors. CONCLUSIONS Our study unveils the parallel evolution of DMG at genetic, epigenetic, and transcriptional levels, unveiling novel subclonal phenotypes governing tumor behavior. Crucially, we identify critical environmental shifts such as hypoxia and inflammatory changes, coupled with specialized signaling programs, driving dissemination and resistance phenotypes. These insights may pave the way for generating accurate genetically mouse models and targeted therapeutic interventions to combat this challenging malignancy.

A practical approach for anabolic treatment of bone fragility with romosozumab.

Romosozumab, a fully humanized anti-sclerostin-antibody, is a bone-builder stimulating osteoblasts and inhibiting osteoclast by activation of the canonical Wnt-beta catenin signaling. This unique mechanism of action has the potential to address unmet needs in osteoporosis management. The multifaceted practical clinical issues related to romosozumab are discussed, especially focusing on the rationale of employing a sclerostin inhibitor to target bone fragility as first line or second line treatment in post-menopausal osteoporosis and in males at increased risk of fractures. Four randomized clinical trials with several post-hoc analyses and more than ten observational studies have consistently demonstrated that romosozumab is effective in rapidly increasing bone mineral density (BMD) and decreasing risk of vertebral, non-vertebral and hip fractures in post-menopausal women at very-high risk of fractures. In male osteoporosis, only data on BMD are available. Noteworthy, romosozumab was shown to be more effective and rapid than teriparatide in improving BMD, bone structure and strength at the hip, especially in women already treated with anti-resorptive drugs. Interestingly, even if romosozumab displays best results in treatment-naïve patients, its favourable effects on BMD were observed even in women previously treated with teriparatide or denosumab, although to a lesser extent. Based on the available evidence, romosozumab could be proposed as ideal drug in several clinical settings, such as non-fractured post-menopausal women at very-high risk of fractures, patients with recent hip fracture, patients non responder to bisphosphonates and short-term denosumab therapy.

Abstract 69: Versican proteolysis signature as a potential immune infiltration biomarker for colorectal cancer

Abstract Background: A subset of patients with mismatch repair proficient colorectal cancers (CRCs) benefit from immunotherapies, though currently no markers predict which cancers are likely to respond. Our group demonstrated that versican (VCAN), an immunoregulatory tumor matrix proteoglycan, when proteolyzed to an immunostimulatory fragment called versikine (Vkine) results in enhanced CD8+ T cell infiltration across multiple cancers. Here we evaluate if a RNA expression signature of VCAN proteolysis can identify CRCs with an immune cell infiltrated phenotype. Methods: The TCGA PanCancer Atlas colorectal adenocarcinoma dataset (594 patients (pts)) was divided into cohorts based on RNA expression Z-scores for VCAN, TIMP3, and ADAMTS4. The VCAN proteolytic predominant (VPP) cohort (Z-score VCAN >0, TIMP3 <0.5, ADAMST4 >0) was compared to the VCAN proteolytic weak (VPW) cohort (Z-score VCAN >0, TIMP3 >0.5, ADAMST4 >0). Differences in clinical characteristics, mutation profile, differential gene expression, and gene set enrichment analysis (GSEA) were evaluated. Results: The VPP group consisted of 42 pts (20:22 female: male, median age: 68.5 years old). The VPW group consisted of 29 pts (12:17 female: male, median age: 64 years old). The two groups had no significant difference between stages of disease. There was a significant increase in the mutation count in the VPP cohort (VPP median=115.5, VPW median=91, p=0.045). The MSI MANTIS scores were not different between the cohorts (VPP median=0.35, VPW median=0.37, p=0.325). The VPP cohort was enriched for gene sets associated with immune cell infiltration (interferon gamma response (FDR q<0.001), inflammatory response (FDR q<0.001), complement (FDR q<0.001), TNFA signaling via NKFB (FDR q<0.001), IL2 STAT5 signaling (FDR q<0.001), and IL6 JAK STAT3 signaling (FDR q<0.001). The VPW cohort was enriched for gene sets involving cell proliferation (WNT beta catenin signaling (FDR q<0.214), hedgehog signaling (FDR q<0.190), and KRAS signaling DN (q<0.159). Specifically, the VPP group demonstrated enhanced expression of CD274 (PD-L1; log2FC=1.41, p<0.001), LAG3(log2FC=1.41, p<0.001), PDF1(log2FC=1.34, p<0.001), CD8A(log2FC=1.25, p<0.001), CXCL9(log2FC=1.19, p=0.001), CXCL10(log2FC=1.44, p=0.001), IL6(log2FC=2.23, p<0.001), and CD19(log2FC=1.26, p<0.001). Conclusion: VPP expression signature correlates with immune infiltration based on RNA expression and deserves further investigation to predict response to immunotherapies in CRC as well as other cancer types. Citation Format: Derek Buckalew, Sean Kraus, Katie Johnson, Cheri A. Pasch, Dustin A. Deming. Versican proteolysis signature as a potential immune infiltration biomarker for colorectal cancer [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 69.

Abstract 7363: Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. BRAF, a protein kinase of the Mitogen-Activated Protein Kinase (MAPK) pathway, is mutated in 12% of colorectal cancers and is a poor prognostic factor for patients. BRAF mutations at the V600 codon are most studied but non-V600 BRAF mutations also exist and behave differently. BRAF V600 CRC treated with BRAF+MEK inhibitors show 12% response rates while we observe 65% response rates in BRAF V600 Melanoma. Additionally, no approved targeted therapies exist for non-V600 BRAF mutant cancers and retrospective studies show poorer PFS with BRAF+MEK inhibitors vs BRAF V600 cancers. We interrogated publicly available data of BRAF mutant CRC to uncover differences in the tumor microenvironment and signalling pathways of BRAF V600 and non-V600 tumors in hopes of uncovering better treatments. Methods: We analyzed the clinical and genomic profile of BRAF mutant CRC tumors from the GENIE v12 data. Gene Set Enrichment Analysis was performed on RNAseq from the TCGA data (discovery cohort) and the combined data from CPTAC and Sidra-LUMC data (validation cohort). We investigated the composition of tumor infiltrating immune cells using CIBERSORTx (deconvolution algorithm for bulk RNAseq). Results: Non-V600 mutations (n=219/1061) were most represented in patients of Asian or Black race compared to White patients (p<0.0001) and importantly, were found in younger patients (p<0.0001). We identified n=67 and n=74 BRAF mutant RNAseq samples in the discovery and validation cohorts, respectively. In the discovery cohort: BRAF V600 CRC were enriched for Interferon Gamma Signaling, Complement pathway, and IL6 JAK STAT3 Signaling (p<0.05) while the non-V600 BRAF mutant CRC showed an enrichment for Wnt-beta Catenin, Notch, and Hedgehog Signaling pathways (p<0.05). In the validation cohorts these same pathways were enriched in V600 and non-V600 BRAF mutant tumors. Interestingly, enrichment of genes co-mutated (GENIE data) in non-V600 BRAF mutant CRC also revealed the Wnt pathway. Non-V600 CRC tumors have a higher proportion of M0 macrophages (p=0.0035) and CD4 memory resting T cells (p<0.0001) while patients with V600 CRC have higher proportions of CD8 T cells (p=0.006) and activated mast cells (p=0.0341). Conclusion: No optimal therapeutic strategies have been defined for these potentially actionable non-V600 BRAF mutant CRCs. By exploring the multi-omic landscape of these tumors we have identified unique characteristics of non-V600 BRAF mutant CRC that could be therapeutically exploited. Citation Format: Emmanuelle Rousselle, Suzanne Kazandjian, April Rose. Clinical, genomic, transcriptomic and immunologic profile of BRAF mutant colorectal tumors [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 7363.

Prognostic significance of LRPPRC and its association with immune infiltration in liver hepatocellular carcinoma.

Leucine rich pentatricopeptide repeat containing (LRPPRC) protein is a multifunctional protein involved in cell cycle progression and tumor development. However, its prognostic significance and association with immune infiltration in Liver hepatocellular carcinoma (LIHC) remain unclear. We utilized transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) databases of LIHC patients to investigate the potential pro-cancer role of LRPPRC, including differential expression of LRPPRC in LIHC, prognostic value, clinicopathological features, immune cell infiltration relevance and function enrichment analysis. Our findings suggest that LRPPRC is upregulated in LIHC and exhibits correlations with survival, clinical stage, and tumor grade in LIHC patients. Additionally, immune infiltration analysis revealed significant negative correlations between LRPPRC expression and multiple tumor-infiltrating immune cells, including CTLs, DCs, pDCs, B cells, Th17 cells, neutrophils, T cells, Mast cells, Th1 cells, Tregs, and NK cells, whereas a significant positive correlation was observed with infiltration of Th2 cells, T helper cells and Tcms. Furthermore, functional enrichment analysis indicated that LRPPRC may be involved in G2m checkpoint, mitotic spindle, E2f targets, Wnt Beta catenin signaling, spermatogenesis and other processes.

Pan-cancer analysis of homeodomain-containing gene C10 and its carcinogenesis in lung adenocarcinoma.

We found elevated homeodomain-containing gene C10 (HOXC10) showed dual roles in cancers' prognosis. Some signal pathways associated with tumor were totally positively enriched in HOXC10 for whole cancers. On the contrary, Notch signaling, Wnt-beta catenin signaling, myogenesis, and Hedgehog signaling were almost negatively enriched in HOXC10. Some pathways showed dual roles such as Kras signaling, interferon gram and alpha response, IL6/JAK/STAT3, IL2/STAT5 signaling. HOXC10 was associated with tumor mutation burden and microsatellite instability. HOXC10 also was associated with tumor microenvironment and immune status. HOXC10 was negatively associated with immune score in most cancers except colon adenocarcinoma. The correlations of HOXC10 with immune-related genes presented dual roles in different cancers. Results from our clinical samples indicated that HOXC10 was an independent predictor for distant metastasis-free survival in lung adenocarcinoma (LUAD). Notably, the high levels of HOXC10 were positively correlated with the expression of angiogenic markers, vascular endothelial growth factor and microvessel density, and the number of CTC clusters. Our results demonstrated that aberrant expression happened in most cancers, which also affected the clinical prognosis and involved in progression via multiple signal pathways cancers. HOXC10 overexpression plays an important role in the aggression and metastasis in LUAD, which indicated a potential therapeutic target and an independent factor for the prognosis for LUAD patients.

HDAC-Mediated Glial Crosstalk Mediates Radiation Induced Memory Changes from Whole Brain Radiation in a Mouse Model

Prior work demonstrated radiation sensitivity can be regulated via Class III HDAC-mediated intracellular DNA damage response coordinated with Wnt-beta catenin signaling. We hypothesized that radiation induced functional alterations in HDAC mediated glial crosstalk in the CNS produce intercellular signaling alterations leading to phenotypic changes in cognition following whole brain radiation exposure. Primary human astrocytes, human astrocytoma (U251), and immortalized human microglia (HMC3) were examined in vitro in response to treatment with conditioned media isolated from irradiated glial cells. Prior to treatment with either single-dose or fractionated radiation schedules, cells were transfected with siRNA expression vector for Sirt2, a class III HDAC, versus scrambled controls. Quantitative PCR and immunoblots for neurotransmitters, metabolism, and inflammatory markers were obtained. Cells exposed to conditioned media were examined by immunofluorescence for cytoskeletal alterations and changes in junctional proteins. The findings were correlated with in vivo qPCR and immunohistochemical changes in brain tissue and functional memory changes in Sirt2 knockout versus wild type mice after whole brain radiation using novel object recognition testing at 2 weeks and 6 month post radiation timepoints. Statistical analysis was performed using paired Students T test between treated and control groups. Cells treated with conditioned media produced from irradiated U251, astrocytes and microglia produced increased expression of inflammatory cytokines IL-6, IL-8, GM-CSF and VEGF. However, immunofluorescence of cells treated with conditioned media from irradiated Sirt2 knockdown microglia and primary astrocytes demonstrated qualitative disruptions in glutamate neurotransmitter metabolism and actin cytoskeletal alterations with changes in pseudopodia and lamellipodia after staining with phalloidin and neurofilament L, suggesting altered intercellular communication. Connexin 43 in gap junctions was increased >2-fold (p<0.05) after exposure to conditioned media, whereas E-cadherin in adherens junctions was not significantly affected. Novel object recognition testing of Sirt2 knockout mice demonstrated resistance to radiation induced memory decline from whole brain radiation at pre radiation day 5 versus post radiation days 5 and 185 compared to controls (p<0.05). Glial crosstalk can be mediated via elimination of a Class III HDAC and appears to be a key mediator of radiation induced disruptions of intercellular communication in the CNS, connecting radiation to structural changes on the cell surface to synaptic activity and neurotransmitter metabolism, leading to functional disputations in recognition memory similar to what is experienced by patients receiving brain radiotherapy. These data suggest glial cross talk as a new therapeutic avenue to combat radiation induced cognitive dysfunction.

Abstract 6074: Germline and somatic genomic profiling of urothelial carcinoma

Abstract Urothelial carcinoma (UC) presents most frequently as bladder cancer and is the most common cancer of the urinary system in the United States. UC relapse and progression are common and impose a significant negative impact on the lives of patients and healthcare resources. To elucidate the biological mechanisms of UC and find novel biomarkers, we analyzed the clinical and genomic data in the Oncology Research Information Exchange Network (ORIEN). We conducted gene-based and gene-set based association tests on rare germline variants comparing the exome sequencing of 336 UC patients and genome sequencing of 366 healthy controls (42 unrelated individuals from the Centre d'Etudes du Polymorphisme Humain [CEPH] families and 324 from the University of Utah Heritage 1000 [H1K] Projects). The analysis of loss-of-function (LoF) variants revealed that the forkhead box (FOX) J2 gene set was significantly associated with UC at the genome-wide level (Bonferroni-corrected p-value=0.01). Genes in this gene set contain a motif that matches the FOXJ2 transcription factor binding site. Firth-penalized Cox proportional hazard regression on overall survival identified LoF variants in genes down-regulated in naive CD8 T cells to be associated with worse prognosis (Bonferroni-corrected p-value=0.032, hazard ratio=28.2, and 95% confidence interval 6.66 to 119.0). In exome sequencing of tumor tissues, we searched for driver genes and pathways by testing for higher variant allelic fractions than the genome average. The tests yielded eleven genes with genome-wide significance (Table 1). By gene set analysis using the MSigDB Hallmark database, significant pathways included the P53 pathway (p&amp;lt;2 × 10−16), Wnt beta-catenin signaling (p&amp;lt;2 × 10−16), E2F targets (p=8 × 10−13), PI3K/AKT/mTOR signaling (4 × 10−7), and apoptosis (p=9 × 10−7). These results reveal the germline predisposition variants and somatic oncogenic drivers in UC and suggest immune evasion as a contributing factor for poor clinical outcomes in UC patients. Table 1. Genes with high allelic fractions Gene P-value Number of Variants TP53 &amp;lt;2 × 10−16 203 RB1 2.42 × 10−16 67 ELF3 2.06 × 10−7 38 TSC1 4.05 × 10−7 32 KMT2D 7.59 × 10−7 91 ZFP36L1 8.80 × 10−7 33 CDKN1A 2.47 × 10−6 40 FGFR3 1.11 × 10−5 37 ARID1A 1.28 × 10−5 62 SMARCA4 1.53 × 10−5 17 PIK3CA 2.00 × 10−5 53 Citation Format: Bing-Jian Feng, Wendy Kohlmann, David A. Nix, Aaron Atkinson, Kenneth M. Boucher, Courtney Carroll, Jill Kolesar, Eric A. Singer, Stephen B. Edge, Kamal Sahu, Alejandro Sanchez, Mikaela Larson, Michelle L. Churchman, Laura Graham, John D. Carpten, Yousef Zakharia, Lindsey Byrne, Rohit K. Jain, Kenneth G. Nepple, Ahmad Shabsigh, Jad Chahoud, Sumati Gupta. Germline and somatic genomic profiling of urothelial carcinoma. [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 6074.

Abstract 1409: RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer

Abstract Background: African American men (AAs) have the highest incidence of prostate cancer (PC) and often develop therapeutic resistance leading to high mortality. The androgen receptor (AR) is paramount in the growth and progression of PC, and insufficient blockade of AR leads to therapeutic resistance. Our previous work shows that CYP3A5 facilitates the activation of AR, promoting the transcription of genes supporting PC growth. CYP3A5 is polymorphic, and the wild-type (wt; *1) variant encoding the full-length active protein is expressed in 73% of AAs and only in 5% of non-Hispanic White Americans (NHWA). The race-linked expression of wt-CYP3A5*1 in AAs can lead to hyperactive AR contributing to therapeutic resistance. We performed an RNA sequencing study utilizing samples from AAs and NWHA patients to understand the allelic influence of CYP3A5 on PC severity. Methods: Illumina RNA sequencing was performed using RNA from 13 AA and 12 NHWA prostatic adenocarcinomas. CYP3A5 genotyping separated them into two groups: a) carrying wt-CYP3A5 (*1/*1 and *1/*3; N=14); and b) mutant CYP3A5 (*3/*3; N=11) variants. The samples’ ancestry was confirmed using markers previously described. Differentially expressed genes (DEGs) between wt-*1 and mutant-*3 CYP3A5 carrying groups were identified using DESeq2, at a false discovery rate of adjusted p &amp;lt; 0.05. Alternately we also used the Kallisto method for quantifying the abundances of the transcript from RNA seq data. Results: Aligned RNA reads with the human genome using DESeq2 resulted in the identification of 30 DEGs between the two groups, of these, 14 genes are known to be dysregulated in PC and promote aggressive disease (SEMG2, ZNRF3, CCL18, U3, MT-ND6, SCARNA9, NRCAM, LINC00853, TCF4, SLC38A6, HHLA3, DECR1, THTPA, and NEFH). Aberrant TCF4 along with ZNRF3 is known to promote PC growth via Wnt-beta catenin signaling. SLC38A6 is a glutamine transporter and is often upregulated in PC to promote glutamine uptake in an AR-dependent manner. NEFH is an intermediate filament protein that is downregulated in PC metastasis. Kallisto method identified 84 DEGs, 11 of those genes overlap with the previous DFGs identified using the transcriptome alignment method. The additional DFGs identified with the Kallisto method include aberrant expression of multiple genes (MT-CO1/2, MT-ND1/6) involved in mitochondrial dysfunction known to contribute towards cell growth and tumorigenesis beyond the Warburg effect. The wt-*1 CYP3A5 expressing group also shows increased expression of several pseudogenes, dysregulation of pseudogenes has been observed in several cancers and can act as a master regulator for gene expression, promoting tumorigenesis. Conclusion: The presence of wt-*1 CYP3A5 may be one of the factors leading to aggressive PC in AAs as it shows differential expression of several genes known to accelerate PC growth. Citation Format: Jeetesh Sharma, Richard Tillett, Shirley Shen, Jabril Johnson, Rick A. Kittles, Mohammad Saleem Bhat, Oscar Goodman Jr, Edwin C. Oh, Ranjana Mitra. RNA sequencing reveals that African Americans carrying wild-type CYP3A5 differentially express genes known to promote aggressive prostate cancer [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 1409.

Comprehensive Explorations of CCL28 in Lung Adenocarcinoma Immunotherapy and Experimental Validation.

Chemokines have been reported to play an important role in cancer immunotherapy. This study aimed to explore the chemokines involved in lung cancer immunotherapy. All the public data were downloaded from The Cancer Genome Atlas Program database. Quantitative real time-PCR was used to detect the mRNA level of specific molecules and Western blot was used for the protein level. Other experiments used include luciferase reporter experiments, flow cytometric analysis, Chromatin immunoprecipitation assay, ELISA and co-cultured system. We found that the CCL7, CCL11, CCL14, CCL24, CCL25, CCL26, CCL28 had a higher level, while the CCL17, CCL23 had a lower level in immunotherapy non-responders. Also, we found that immunotherapy non-responders had a higher level of CD56dim NK cells, NK cells, Th1 cells, Th2 cells and Treg, yet a lower level of iDC and Th17 cells. Biological enrichment analysis indicated that in the patients with high Treg infiltration, the pathways of pancreas beta cells, KRAS signaling, coagulation, WNT BETA catenin signaling, bile acid metabolism, interferon alpha response, hedgehog signaling, PI3K/AKT/mTOR signaling, apical surface, myogenesis were significantly enriched in. CCL7, CCL11, CCL26 and CCL28 were selected for further analysis. Compared with the patients with high CCL7, CCL11, CCL26 and CCL28 expression, the patients with low CCL7, CCL11, CCL26 and CCL28 expression had a better performance of immunotherapy response and this effect might partly be due to Treg cells. Furthermore, biological exploration and clinical correlation of CCL7, CCL11, CCL26 and CCL28 were conducted, Finally, CCL28 was selected for validation. Experiments showed that under the hypoxia condition, HIF-1α was upregulated, which can directly bind to the promoter region of CCL28 and lead to its higher level. Also, CCL28 secreted by lung cancer cells could induce Tregs infiltration. Our study provides a novel insight focused on the chemokines in lung cancer immunotherapy. Also, CCL28 was identified as an underlying biomarker for lung cancer immunotherapy.

Expression profiling suggests the involvement of hormone-related, metabolic, and Wnt signaling pathways in pterygium progression.

BackgroundPterygium is an ocular surface disease that can cause visual impairment if it progressively invades the cornea. Although many pieces of research showed ultraviolet radiation is a trigger of pterygium pathological progress, the underlying mechanism in pterygium remains indistinct.MethodsIn this study, we used microarray to evaluate the changes of transcripts between primary pterygium and adjacent normal conjunctiva samples in China. Then, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. Moreover, we constructed protein-protein interaction (PPI) and miRNA-mRNA regulatory networks to predict possible regulatory relationships. We next performed gene set enrichment analysis (GSEA) to explore the similarities and differences of transcripts between Asian studies from the Gene Expression Omnibus database. Furthermore, we took the intersection of differentially expressed genes (DEGs) with other data and identified hub genes of the development of pterygium. Finally, we utilized real-time quantitative PCR to verify the expression levels of candidate genes.ResultsA total of 49 DEGs were identified. The enrichment analyses of DEGs showed that pathways such as the Wnt-signaling pathway and metabolism-related pathways were upregulated, while pathways such as hormone-related and transcription factor-associated pathways were downregulated. The PPI and miRNA-mRNA regulatory networks provide ideas for future research directions. The GSEA of selecting Asian data revealed that epithelial-mesenchymal transition and myogenesis existed in the pathology of pterygium in the Asian group. Furthermore, five gene sets (interferon-gamma response, Wnt beta-catenin signaling, oxidative phosphorylation, DNA repair, and MYC targets v2) were found only in our Chinese datasets. After taking an intersection between selecting datasets, we identified two upregulated (SPP1 and MYH11) and five downregulated (ATF3, FOS, EGR1, FOSB, and NR4A2) hub genes. We finally chose night genes to verify their expression levels, including the other two genes (SFRP2 and SFRP4) involved in Wnt signaling; Their expression levels were significantly different between pterygium and conjunctiva.ConclusionsWe consider hormone-related, metabolic, and Wnt signaling pathways may be important in the pathology of pterygium development. Nine candidate genes we identified deserve further study and can be potential therapeutic targets.

Sphingolipid metabolism is associated with osteosarcoma metastasis and prognosis: Evidence from interaction analysis.

BackgroundMetabolism is widely involved in the occurrence and development of cancer. However, its role in osteosarcoma (OS) has not been elucidated.MethodsThe open-accessed data included in this study were downloaded from The Cancer Genome Atlas (TCGA) database (TARGET-OS project). All the analysis was performed in R environments.ResultsBased on the single sample gene set enrichment analysis algorithm, we quantified 21 metabolism terms in OS patients. Among these, sphingolipid metabolism was upregulated in the metastatic OS tissue and associated with a worse prognosis, therefore aroused our interest and selected for further analysis. Our result showed that sphingolipid metabolism could activate the Notch signaling and angiogenesis pathway, which might be responsible for the metastasis ability and poor prognosis. A protein-protein interaction network was constructed to illustrate the interaction of the differentially expressed genes between high and low sphingolipid metabolism. Immune analysis showed that multiple immune terms were upregulated in patients with high sphingolipid metabolism activity. Then, a prognosis model was established based on the identified DEGs between patients with high and low sphingolipid metabolism, which showed great prediction efficiency. Pathway enrichment showed the pathway of myogenesis, spermatogenesis, peroxisome, KRAS signaling, pancreas beta cells, apical surface, MYC target, WNT beta-catenin signaling, late estrogen response and apical junction was significantly enriched in high risk patients. Moreover, we found that the model genes MAGEB1, NPIPA2, PLA2G4B and MAGEA3 could effectively indicate sphingolipid metabolism and risk group.ConclusionsIn summary, our result showed that sphingolipid metabolism is associated with osteosarcoma metastasis and prognosis, which has the potential to be a therapeutic target for OS.

Identification of the alpha linolenic acid metabolism-related signature associated with prognosis and the immune microenvironment in nasopharyngeal carcinoma.

BackgroundTumor metabolism is important for cancer progression. Nevertheless, the role of the metabolism pathway and related molecules in nasopharyngeal carcinoma (NPC) is limited.MethodsOpen-accessed data was downloaded from The Cancer Genome Atlas database. All the analysis was performed using the R software and the package in R environments.ResultsIn our study, we firstly explored the role of 21 metabolism-related pathways in NPC patients. We found that the steroid biosynthesis and biosynthesis of unsaturated fatty acids were risk factors, while the alpha linolenic acid metabolism was a protective factor. Then, the alpha linolenic acid metabolism aroused our interest. A total of 128 differentially expressed genes (DEGs) were identified, including 71 downregulated and 57 upregulated genes identified between high and low alpha linolenic acid metabolism level. Based on these DEGs, we constructed a prognosis model including DEFB4B, FOXL2NB, MDGA2, RTL1, SLURP2, TMEM151B and TSPAN19, which showed great prediction efficiency in both training and validation cohorts. Clinical correlation analysis showed that high-risk patients might have worse clinical pathology parameters. Pathway enrichment analysis showed that riskscore was positively correlated with angiogenesis, DNA repair, G2/M checkpoints, IL6/JAK/STAT3 signaling, KRAS signaling up, WNT beta-catenin signaling, PI3K/AKT/mTOR signaling, yet positively correlated with inflammatory response, xenobiotic metabolism, TNF-α signaling via NFKB and interferon-gamma response. Immune infiltration analysis showed that the riskscore was positively correlated with the M2 and M0 macrophages, but negatively correlated with neutrophils, plasma cells, follicular helper T cells and resting dendritic cells Moreover, we found that the low-risk patients might be more sensitive to immunotherapy and lapatinib.ConclusionsIn all, our study identified the genes associated with alpha linolenic acid metabolism and constructed an effective prognosis model which could robustly predict NPC patients prognosis.

REMOVED: MiR-539-3p inhibits osteoblast activity and bone formation in vivo via targeting LRP-6

X-linked hypophosphatemia (XLH), the most common form of inherited rickets, is characterized by renal phosphate (Pi) wasting, abnormal vitamin D metabolism and defective bone mineralization. It is caused by an inactivation mutation in PHEX gene. Other factors, such as microRNA (miRNA), which regulates biological functions by interfering with specific mRNA translation, also contribute in controlling disease pathogenesis. Recent studies indicate the role of miRNAs in osteoblast functions. The main objective of this study is to identify novel miRNAs, regulated by Phex in osteoblast cells, and to determine whether miR-539-3p, a known tumor suppressor, aids in osteogenesis. MiRNA expression patterns in control and siPhex treated cells were analyzed by miRNA microarray profiling and quantitative RT-PCR. Effect of miR-539-3p on osteoblast differentiation was achieved by transfection with miR-539-3p and anti-miR in mouse calvarial osteoblasts. MiR-539-3p targets were identified by Luciferase reporter gene assay. LRP-6, Wnt3, Beta catenin, p-beta catenin, GSK3β and Lef-1 protein levels were determined by Western blotting. To investigate the function of miR-539-3p in vivo, animal groups were divided into; sham+PBS (ovary intact), ovariectomized (Ovx)+miC, Ovx+miR and Ovx+anti-miR. Chemically modified oligonucleotide miR and anti-miR were injected at 5ug/animal dose via a subcutaneously injection into mice. In PHEX silenced mice calvarial osteoblast cells, miR-539-3p was up-regulated as analyzed by miRNA array profiling and qRT-PCR analysis. Over-expression of miR-539-3p inhibited osteoblast differentiation, whereas inhibition of miR-539-3p function promoted expression of osteoblast-specific genes and alkaline phosphatase (ALP) activity. Target prediction analysis tools and experimental validation by luciferase 3ⲠUTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over-expression of miR-539-3p led to LRP-6 repression and inhibition of Wnt-Beta catenin signaling. Furthermore, miR-539-3p silencing led to increased bone formation and improved trabecular micro architecture in Ovx mice. Although miR-539-3p is known to be a tumor repressor, we identified a second complementary function of miR-539-3p where it inhibits LRP-6-mediated osteogenesis. Our findings suggest that pharmacological inhibition of miR-539-3p by anti-miR-539-3p could represent a therapeutic strategy for enhancing bone formation in vivo.

A Review of the Pathophysiological Mechanisms Underlying Castration-resistant Prostate Cancer.

Androgen deprivation therapy or ADT is one of the cornerstones of management of locally advanced or metastatic prostate cancer, alongside radiation therapy. However, despite early response, most advanced prostate cancers progress into an androgen unresponsive or castrate resistant state, which hitherto remains an incurable entity and the second leading cause of cancer-related mortality in men in the US. Recent advances have uncovered multiple complex and intermingled mechanisms underlying this transformation. While most of these mechanisms revolve around androgen receptor (AR) signaling, novel pathways which act independently of the androgen axis are also being discovered. The aim of this article is to review the pathophysiological mechanisms that help bypass the apoptotic effects of ADT to create castrate resistance. The article discusses castrate resistance mechanisms under two categories: 1. Direct AR dependent pathways such as amplification or gain of function mutations in AR, development of functional splice variants, posttranslational regulation, and pro-oncogenic modulation in the expression of coactivators vs corepressors of AR. 2. Ancillary pathways involving RAS/MAP kinase, TGF-beta/SMAD pathway, FGF signaling, JAK/STAT pathway, Wnt-Beta catenin and hedgehog signaling as well as the role of cell adhesion molecules and G-protein coupled receptors. miRNAs are also briefly discussed. Understanding the mechanisms involved in the development and progression of castration-resistant prostate cancer is paramount to the development of targeted agents to overcome these mechanisms. A number of targeted agents are currently in development. As we strive for more personalized treatment across oncology care, treatment regimens will need to be tailored based on the type of CRPC and the underlying mechanism of castration resistance.

O18 Integrated molecular analysis of systemic sclerosis skin and blood shows significant differences between major autoantibody subgroups

Abstract Background/Aims The major antinuclear autoantibodies of systemic sclerosis (SSc) associate with different skin score trajectories and risk of internal organ manifestations. To elucidate molecular differences between ANA-defined subgroups, we utilised the prospective BIOPSY cohort of well-characterised SSc patients. Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc, 16 lcSSc) and 16 healthy controls (HC). 36 (69%) of the SSc patients are female. Mean disease duration in the early dcSSc cohort was 24 months (sd 12 months), and in established dcSSc was 11.3 years. ANA frequency in BIOPSY reflected the overall dcSSc population: anti-topoisomerase-1 (ATA) n = 14 (27%), anti-RNA pol III (ARA) n = 12 (23%) and other n = 26 (50%). Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). At a group level mRSS peak was 21.9 (11.8) at 3 months and fell to 19.1(10.5) at 12 months. Serum biomarkers of ECM turnover and fibrosis were measured three monthly and genome-wide transcriptomic profiling of whole skin and whole blood performed by RNA-Seq. Statistical analysis used RStudio with ANOVA, and Tukey post-hoc test. Differential gene expression used the Bioconductor limma software, with standard thresholds for significance. Results At baseline, there were differences in soluble markers between clinical SSc sugroups and HC but not for major ANA subgroups. However, we found clear differences in early dcSSc analysed by major ANA subset for longitudinal change in serum markers of fibrosis and in whole skin gene expression, suggesting a mechanistic basis for the distinct clinical phenotypes associated with hallmark ANAs. During follow-up, significant differences were observed in HA, TIMP1, and PIIINP at 6 and 12 months (p &amp;lt; 0.05), with stable levels in ATA+ patients compared to progressively increased levels in the other subgroups. There were 564 significantly differentially expressed genes in skin between early dcSSc and HC. Unsupervised clustering differentiated patients with ARA and ATA positivity with early dcSSc. 54 genes were significantly differentially expressed in skin between ATA and ARA patients. Whilst 179 genes were differentially expressed in whole blood between early dcSSc compared with HC, no genes could significantly differentiate ATA from ARA. Functional analysis using HALLMARK pathway analysis identified both shared pathways associated with SSc across ANA groups (e.g. TGF beta signaling, IL6 JAK STAT3 signalling, inflammatory response), and pathways only upregulated in patients with ATA (e.g. Wnt beta catenin signaling, Notch signaling), and ARA (e.g. interferon gamma response, adipogenesis). Conclusion We have found significant differences in skin gene expression and longitudinal change in serum markers by autoantibody specificity in dcSSc. Our findings have implications for SSc pathogenesis and support stratification by ANA subgroup in clinical studies. Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. N. Galwey: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Abstract PS17-41: In depth single cell profiling of a case of bone metastases with associated organoid models reveals a precision medicine approach to treatment

Abstract Occurring in 65-80% of metastatic breast cancer (BC), bone metastasis (BoM) is the major cause of BC related mortality. Under current standard of care, 5 year overall survival for patients with BoM is 20-25%, warranting the need of improved treatments. Like other cancers, BC BoM has high inter- and intra-patient heterogeneity. Thus, understanding disease evolution and heterogeneity of BoM at the individual patient level will be key to guide precise application of targeted therapies. In this study, we describe in depth histologic and single cell molecular characterization of a case of invasive lobular breast cancer (ILC) metastasis to bone. We collected fixed primary tumor (ER+/PR-/HER2- ILC) and fresh pelvis and tibia BoMs from a patient. H&amp;E/IHC staining and whole exome sequencing (WES) of DNA and RNA of the samples were performed. Organoids were derived from the two BoMs and single cell RNA sequencing was applied to these cultures and the BoMs. Based on H&amp;E/IHC staining, this case evolved from a ER+ primary ILC to ER- BoM with mixed lobular and ductal carcinoma features. WES revealed 208 missense somatic mutations in the BoMs. 15 were predicted as BC drivers, two of which are druggable including PIK3CA (E545K) and BRCA1 (D1834H, H399P). Notably, BRCA1 (D1834H) was not found in the primary tumor. Based on RNA sequencing, estrogen signaling was downregulated, while TGF-β, Wnt beta catenin and PI3K signaling, epithelial to mesenchymal transition (EMT) and angiogenesis were upregulated, all of which have been reported to be potential targets in BoMs. scRNAseq of BoMs revealed 5 major cell populations and pronounced heterogeneity. The two BoMs showed similar cellular compositions, including epithelial (30-50%), fibroblasts (50%), immune (5-10%), osteoclasts (1-2%) and endothelial cells (1-2%). Within epithelial cells, there were 5 major clusters exhibiting unique transcriptomic features, including a TNF-α signaling high cluster, two clusters showing high partial EMT (pEMT) signatures majorly regulated by PRRX1/2, TWIST1/2, and FOXS1, a cluster in active cell proliferation, and a cluster showing high signatures related to endocrine resistance. In fibroblasts, 3 clusters were identified representing ECM remodeling, angiogenesis (VEGFA high) and myofibroblasts. Immune cells majorly composed of macrophages, CD4+, CD8+ and Treg T cells. 2.2% tumor and 3.2% T cells express PDL1, exceeding the 1% threshold to select patients eligible for anti-PDL1 therapy. Based on CellPhoneDB, epithelial cells with high pEMT signature show the most interactions and uniquely interact with Treg cells through TNFSF4-TNFRSF4 which has been reported to promote Treg cell proliferation, and is investigated in clinic for immune therapy. Organoids were developed to evaluate therapeutic potential of targetable mutations, upregulated genes and susceptible cell populations. WES showed organoids preserved the mutational signatures of the matched tumor. scRNAseq showed all epithelial populations in the tumor were preserved in the matched organoids at comparable abundance. Consistent with the BRCA1 and PIK3CA mutations, organids were responsive to a PARP (Talazoparib: IC50 1.3uM/1.3uM) and PI3K (Alpelisib: IC50 9uM/4uM) inhibitor. We are currently examining the therapeutic potential of inhibiting pEMT, TNF-α, and genes upregulated in BoM. In summary, we have demonstrated a precision medicine approach to understand the evolution and heterogeneity of BC BoM. We have identified potential therapeutic targets and evaluated those in patient-specific organoids, thereby providing insights for the design of a precision medicine based clinical treatment strategy. Citation Format: Kai Ding, Fangyuan Chen, Nolan Priedigkeit, Daniel D. Brown, Tanya Heim, Rebecca Watters, Kurt Weiss, Jennifer Atkinson, Steffi Oesterreich, Adrian Lee. In depth single cell profiling of a case of bone metastases with associated organoid models reveals a precision medicine approach to treatment [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-41.

The Mutational and Transcriptional Landscapes of Hepatocarcinogenesis in a Rat Model.

SummaryHepatocellular carcinoma (HCC) initiation is characterized by stepwise accumulation of molecular alterations, during which the early events are largely unknown. Here, we presented a comprehensive genomic and transcriptomic landscape at stages of hepatitis, cirrhosis, and HCC by using a diethylnitrosamine-induced rat HCC model. We observed the early occurrence of gene instability and aberrant cancer associated signaling pathways in liver hepatitis. We further characterized the progressive molecular changes during hepatocarcinogenesis, wherein the intense rivalry between tumor-suppressive and oncogenic strengths occurred in cirrhosis stage. Despite the significant pathological difference, mutation signatures and expression landscape are highly similar between hepatitis and cirrhosis stages. Furthermore, we identified PI3K-Akt signaling pathway as a key pathway in the process of hepatocarcinogenesis through integrative analysis, and PIK3CD is a potential biomarker indicating HCC recurrence. The dynamic immune response during hepatocarcinogenesis, such as continuous decline of monocytes, suggests an immunological intervention strategy beyond chemoprevention for liver cancer.

3033 – SINGLE-CELL MULTI-OMIC ANALYSIS UNRAVELS GENETIC AND MOLECULAR HETEROGENEITY IN TRANSFORMED MYELOPROLIFERATIVE NEOPLASMS

One out of five patients with myeloproliferative neoplasms (MPN) will progress to an aggressive and largely untreatable form of Acute Myeloid Leukaemia (post-MPN secondary AML, or sAML). Transformation is associated with acquisition of TP53 missense mutations in approximately 50% of cases, and markedly increased genetic and molecular heterogeneity. This makes it challenging to effectively eliminate all subpopulations of tumour cells, and likely underlies the mostly universal treatment failure in sAML. Characterizing the complex genetic and cellular hierarchies associated with TP53 transformation is crucial for the development of effective targeted therapies. To resolve these multiple layers of intratumoral heterogeneity, we recently developed TARGET-seq (Rodriguez-Meira et al, 2019), a single-cell multi-omic method that correlates genetic, transcriptional and cell-surface proteomic readouts from the same single cell with extremely high resolution. TARGET-seq analysis of over 7000 Lineage-CD34+ cells (indexed for CD38/90/45RA/123) from TP53-mutant sAML patients revealed striking patterns of clonal evolution in different immunophenotypically defined stem/progenitor cell types, and identified preleukemic and leukemic subclones with markedly distinct molecular signatures. Acquisition of TP53 mutations was invariably associated with highly complex clonal evolution, enrichment of cell-cycle associated transcription and downregulation of key stem cell regulators. Interestingly, preleukemic subclones from patients undergoing transformation were highly enriched in WNT-beta catenin signaling and depleted in DNA-repair genes, as compared to non-transformed MPN patients. This suggests that preleukemic clones could be primed for disease transformation, and could potentially be targeted to prevent disease progression. Ultimately, single-cell multi-omic analysis identified key molecular regulators of TP53 mediated transformation, providing unique insights into the evolution of these tumors and how they might be therapeutically targeted.