Metastatic Gene Research Articles

P60.03 Transcriptome and Genome Profiles of Metastatic Lung Cancers Highlighted Predictive Power of Infection-Related Model in Metastasis

Metastasis seriously affected the prognosis of lung cancer. The close relationship between microbes and lung cancer was found. However, the roles of infection-related pathways and genes in lung cancer metastasis remained unclear.

253 Targeting tumour vascularisation through endothelial-specific Sox9 deletion reduces melanoma metastasis through reduction of vascularisation, changing melanoma gene expression pattern

The formation of de novo vascular structures is vital for melanoma growth and metastasis. Endovascular progenitor cells (EVPs) residing in vessel walls give rise to mature endothelial cells and contribute to a new blood vessel network formation in tumours. Sox9 is a transcription factor that is highly upregulated in EVPs and is playing a crucial role in stem cell self-renewal and quiescence. In this study, we aimed to explore the role of Sox9 deletion in the endothelium on tumour vascularisation, metastasis, and melanoma gene expression. An endothelial-specific knock out mouse model, Sox9fl/fl/Cdh5CreERt2/Rosa-YFP was utilised to delete Sox9 conditionally in the endothelium. A significant reduction in tumour EVPs was observed upon Sox9 deletion in the endothelium in B16-F0 or HcMel12 melanoma tumours. Immunofluorescence staining of tumour sections demonstrated a significant reduction in the number and area of CD31+ vessels and a significant increase of pericytes coverage suggesting increased maturity of the remaining vessels. Lung metastasis significantly reduced after surgical excision of the primary Hcmel12 tumours. Of importance, intravenous delivery of B16-F10 melanoma cells engrafting and forming lung metastases was not changed by Sox9 deletion in the endothelium suggesting that Sox9 deletion acts primarily in the primary tumour. Deletion of Sox9 in the vasculature led to significant gene expression changes in flow-sorted melanoma cells in the primary tumour. Epithelial to mesenchymal transition pathway was among the downregulated pathways suggesting that deletion of Sox9 in the endothelium affected the ability of primary tumour cells to metastasize. In summary, Sox9 deletion in the endothelium resulted in the depletion of EVPs, prompting fewer de novo vessels in the tumour and reduced metastasis. This new knowledge strongly suggests EVPs as a new target in the field of anti-vascularisation treatment in cancer and metastasis.

Characterization and prognostic value of LXR splice variants in triple-negative breast cancer

SummaryActivity of liver x receptor (LXR), the homeostatic regulator of cholesterol metabolism, is elevated in triple-negative breast cancer (BCa) relative to other BCa subtypes, driving drug resistance and metastatic gene signatures. The loci encoding LXRα and LXRβ produce multiple alternatively spliced proteins, but the true range of variants and their relevance to cancer remain poorly defined. Here, we report seven LXR splice variants, three of which have not previously been reported and five that were prognostic for disease-free survival. Expression of full-length LXRα splice variants was associated with poor prognosis, consistent with a role as an oncogenic driver of triple-negative tumor pathophysiology. Contrary to this was the observation that high expression of truncated LXRα splice variants or any LXRβ splice variant was associated with longer survival. These findings indicate that LXR isoform abundance is an important aspect of understanding the link between dysregulated cholesterol metabolism and cancer pathophysiology.

PRAMEF2-mediated dynamic regulation of YAP signaling promotes tumorigenesis

PRAMEF2 is a member of the PRAME multigene family of cancer testis antigens, which serve as prognostic markers for several cancers. However, molecular mechanisms underlying its role in tumorigenesis remain poorly understood. Here, we report that PRAMEF2 is repressed under conditions of altered metabolic homeostasis in a FOXP3-dependent manner. We further demonstrate that PRAMEF2 is a BC-box containing substrate recognition subunit of Cullin 2-based E3 ubiquitin ligase complex. PRAMEF2 mediates polyubiquitylation of LATS1 kinase of the Hippo/YAP pathway, leading to its proteasomal degradation. The site for ubiquitylation was mapped to the conserved Lys860 residue in LATS1. Furthermore, LATS1 degradation promotes enhanced nuclear accumulation of the transcriptional coactivator YAP, resulting in increased expression of proliferative and metastatic genes. Thus, PRAMEF2 promotes malignant phenotype in a YAP-dependent manner. Additionally, elevated PRAMEF2 levels correlate with increased nuclear accumulation of YAP in advanced grades of breast carcinoma. These findings highlight the pivotal role of PRAMEF2 in tumorigenesis and provide mechanistic insight into YAP regulation.

MYC Levels Regulate Metastatic Heterogeneity in Pancreatic Adenocarcinoma.

The degree of metastatic disease varies widely among patients with cancer and affects clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multifluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC)-a tumor type in which most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor-associated macrophages, leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC. SIGNIFICANCE: Here, we investigate metastatic variation seen clinically in patients with PDAC and murine PDAC tumors and identify MYC as a major driver of this heterogeneity.This article is highlighted in the In This Issue feature, p. 275.

Differential Expression of COL1A1, COL1A2, COL6A3, and SULF1 as Prognostic Biomarkers in Gastric Cancer.

BackgroundGastric cancer (GC) is among the most prevalent cancers globally. As such, there is a need to explore the mechanism underlying its pathogenesis and identify potential biomarkers for its prognosis.MethodsONCOMINE was used to screen differentially expressed genes between GC and normal gastric mucosa. GEPIA was used to analyze the expression and correlation of candidate genes in tumor node metastasis (TNM) stage. STRING was used to construct protein interaction network. Kaplan–Meier plotter was used to analyze survival. TIMER was used to evaluate the association between candidate genes and immune cell infiltration.ResultsFrom the ONCOMINE database, we found COL1A1, COL1A2, COL6A3, and SULF1 genes were significantly upregulated in stomach adenocarcinomas. There was a considerable correlation between the expression of COL1A1 (p = 0.029), COL1A2 (p = 0.004), COL6A3 (p = 0.002), SULF1 (p = 0.001), and the TNM stage. COL1A1 was positively correlated with ERBB2 (R = −0.037, p = 0.46), while the other three genes were negatively correlated with ERBB2 (p > 0.05). The Kaplan–Meier plotter showed that low transcriptional levels of COL1A1 (p = 0.0020), COL1A2 (p = 0.0015), COL6A3 (p = 0.0015), and SULF1 (p = 0.0016) in gastric cancer patients were remarkably related to longer overall survival. In addition, there was a close relationship between chemokine expression and infiltration of the six immune cell types: B cells, macrophages, CD4+ T cells, CD8+ T cells, dendritic cells, and neutrophils, implying that the genes acted as indicators of both prognosis and immune status.ConclusionOur findings implicate COL1A1, COL1A2, COL6A3, and SULF1 as candidate biomarkers for the prognosis of gastric cancer.

In-Silico Analysis of Differentially Expressed Genes and Their Regulating microRNA Involved in Lymph Node Metastasis in Invasive Breast Carcinoma

Axillary nodal metastasis is related to poor prognosis in breast cancer (BC). Key candidate genes in BC lymph node metastasis have been identified from Gene Expression Omnibus datasets and explored through functional enrichment database for annotation, visualization and integrated discovery (DAVID) , protein-protein interaction by Search Tool for the Retrieval of Interacting Genes and proteins (STRING), network visualization (Cytoscape), survival analysis (GEPIA, KM Plotter), and target prediction (miRNet). A total of 102 overlapping differentially expressed genes were found. In-silico survival and expression analyses revealed six candidate hub genes, Desmocollin 3 (DSC3), KRT5, KRT6B, KRT17, KRT81, and SERPINB5, to be significantly associated with nodal metastasis and overall survival, and 83 MicroRNA (miRNAs), which may be potential diagnostic markers and therapeutic targets in BC patients.

Identification of Candidate Biomarkers and Prognostic Analysis in Colorectal Cancer Liver Metastases.

BackgroundColorectal cancer (CRC), one of the most common malignant tumors worldwide, has a high mortality rate, especially for patients with CRC liver metastasis (CLM). However, CLM pathogenesis remains unclear.MethodsWe integrated multiple cohort datasets and databases to clarify and verify potential key candidate biomarkers and signal transduction pathways in CLM. GEO2R, DAVID 6.8, ImageGP, STRING, UALCAN, ONCOMINE, THE HUMAN PROTEIN ATLAS, GEPIA 2.0, cBioPortal, TIMER 2.0, DRUGSURV, CRN, GSEA 4.0.3, FUNRICH 3.1.3 and R 4.0.3 were utilized in this study.ResultsSixty-three pairs of matched colorectal primary cancer and liver metastatic gene expression profiles were screened from three gene expression profiles (GSE6988, GSE14297 and GSE81558). Thirty-one up-regulated genes and four down-regulated genes were identified from these three gene expression profiles and verified by another gene expression profiles (GSE 49355) and TCGA database. Two pathways (IGFBP-IGF signaling pathway and complement-coagulation cascade), eighteen key differentially expressed genes (DEGs), six hub genes (SPARCL1, CDH2, CP, HP, TF and SERPINA5) and two biomarkers (CDH2 and SPARCL1) with significantly prognostic values were screened by multi-omics data analysis and verified by Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) cohort.ConclusionsIn this study, we identified a robust set of potential candidate biomarkers in CLM, which would provide potential value for early diagnosis and prognosis, and would promote molecular targeting therapy for CRC and CLM.

Screening and evaluation of the role of immune genes of brain metastasis in lung adenocarcinoma progression based on the TCGA and GEO databases.

BackgroundBrain metastasis was one of the factors leading to the poor long-term prognosis of patients with lung adenocarcinoma (LUAD).MethodsThe expression levels of immune genes in LUAD and LUAD brain metastases tissues were analyzed in GSE161116 dataset using the GEO2R, and the levels of differential immune genes in normal lung and LUAD tissues were verified. The biological functions and signaling mechanisms of the differential immune genes were explored via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Cox regression analysis was used to screen the prognostic factors of LUAD patients, and a risk model was constructed. The role of the model was checked in the development of LUAD via receiver operating characteristic analysis, gene set enrichment analysis, and Cox regression analysis.ResultsDifferentially expressed genes (DEGs) in brain metastasis were involved in the adaptive immune response, B cell differentiation, leukocyte migration, NF-kB signaling pathway, among others. The expression levels of TNFRSF11A, MS4A2, IL11, CAMP, MS4A1, and F2RL1 were independent factors affecting the poor prognosis of LUAD patients via Cox regression analysis and Akaike information criterion. In the constructed risk model, the overall survival of LUAD patients in the high-risk group was poor. The risk model was significantly related to the gender, clinical stage, T stage, lymph node metastasis, and survival status of LUAD patients. In addition, the risk model score was an independent risk factor that affected the poor prognosis of LUAD patients. TNFRSF11A, CAMP, F2RL1, IL11, MS4A1, and MS4A2 of the risk factors had diagnostic significance in LUAD brain metastasis and LUAD. The risk model participated in cytokinetic process, cell cycle, citrate cycle TCA cycle, etc. The risk model score was correlated with the levels of B cells memory, mast cells resting, macrophages M0, mast cells activated, neutrophils, eosinophils, T cells gamma delta, and immune cell markers.ConclusionsThe risk model based on the LUAD brain metastasis immune factors TNFRSF11A, MS4A2, IL11, CAMP, MS4A1, and F2RL1 was related to the diagnosis, poor prognosis, and immune infiltrating cells of LUAD patients, and is expected to provide a reference for the development of treatment strategies for LUAD patients.

ARA-linker-TGFαL3: a novel chimera protein to target breast cancer cells.

Targeted cancer therapies based on overexpressed receptors and the fractions containing immunotoxins and bacterial metabolites are one of the well-known methods to overcome the chemotherapy resistance of cancer cells. In this paper, we designed ARA-linker-TGFαL3, using Arazyme, a Serratia proteamaculans metabolite, and a third loop segment of TGFα to target EGFR-expressing breast cancer cells. After cloning in pET28a (+), the expression of recombinant protein was optimized in Escherichia coli strain BL21 (DE3). MDA-MB-468 (EGFR positive) and MDA-MB-453 (EGFR negative) breast cancer cell lines were employed. Also, the chemotherapeutic drug, Taxotere (Docetaxel), was employed to compare cytotoxicity effects. Cell ELISA assessed the binding affinity of recombinant proteins to the receptor, and the cytotoxicity was detected by MTT and lactate dehydrogenase release assays. The interfacing with cancer cell adhesion was evaluated. Furthermore, the induction of apoptosis was examined utilizing flow cytometric analysis, and caspase-3 activity assay. Moreover, RT-PCR was conducted to study the expression of apoptosis (bax, bcl2, and casp3), angiogenesis (vegfr2), and metastasis (mmp2 and mmp9) genes. ARA-linker-TGFαL3 revealed a higher binding affinity, cytotoxicity, and early apoptosis induction in MDA-MB-468 cells compared to the effects of Arazyme while both recombinant proteins showed similar effects on MDA-MB-453. In addition, the Taxotere caused the highest cytotoxicity on cancer cells through induction of late apoptosis. Meanwhile, the expression of angiogenesis and metastasis genes was decreased in both cell lines after treatment with either ARA-linker-TGFαL3 or Arazyme. Our in vitro results indicated the therapeutic effect of ARA-linker-TGFαL3 on breast cancer cells.

Comprehensive characterization of distinct genetic alterations in metastatic breast cancer across various metastatic sites

Metastasis is the major cause of death in breast cancer patients. Although previous large-scale analyses have identified frequently altered genes specific to metastatic breast cancer (MBC) compared with those in primary breast cancer (PBC), metastatic site-specific altered genes in MBC remain largely uncharacterized. Moreover, large-scale analyses are required owing to the low expected frequency of such alterations, likely caused by tumor heterogeneity and late dissemination of breast cancer. To clarify MBC-specific genetic alterations, we integrated publicly available clinical and mutation data of 261 genes, including MBC drivers, from 4268 MBC and 5217 PBC patients from eight different cohorts. We performed meta-analyses and logistic regression analyses to identify MBC-enriched genetic alterations relative to those in PBC across 15 different metastatic site sets. We identified 11 genes that were more frequently altered in MBC samples from pan-metastatic sites, including four genes (SMARCA4, TSC2, ATRX, and AURKA) which were not identified previously. ARID2 mutations were enriched in treatment-naïve de novo and post-treatment MBC samples, compared with that in treatment-naïve PBC samples. In metastatic site-specific analyses, associations of ESR1 with liver metastasis and RICTOR with bone metastasis were significant, regardless of intrinsic subtypes. Among the 15 metastatic site sets, ESR1 mutations were enriched in the liver and depleted in the lymph nodes, whereas TP53 mutations showed an opposite trend. Seven potential MBC driver mutations showed similar preferential enrichment in specific metastatic sites. This large-scale study identified new MBC genetic alterations according to various metastatic sites and highlights their potential role in breast cancer organotropism.

Integrative Bioinformatics Study of Tangeretin Potential Targets for Preventing Metastatic Breast Cancer.

Agents that target metastasis are important to improve treatment efficacy in patients with breast cancer. Tangeretin, a citrus flavonoid, exhibits antimetastatic effects on breast cancer cells, but its molecular mechanism remains unclear. Tangeretin targets were retrieved from PubChem, whereas metastatic breast cancer regulatory genes were downloaded from PubMed. In total, 58 genes were identified as potential therapeutic target genes of tangeretin (PTs). GO and KEGG pathway enrichment analyses of PTs were performed using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit). The PPI network was analyzed using STRING-DB v11.0 and visualized by Cytoscape software. Hub genes were selected on the basis of the highest degree score as calculated by the CytoHubba plugin. Genetic alterations of the PTs were analyzed using cBioPortal. The prognostic values of the PTs were evaluated with the Kaplan–Meier plot. The expression of PTs across breast cancer samples was confirmed using GEPIA. The reliability of the PTs in metastatic breast cancer cells was validated using ONCOMINE. Molecular docking was performed to foresee the binding sites of tangeretin with PIK3Cα, MMP9, PTGS2, COX-2, and IKK. GO analysis showed that PTs participate in the biological process of stimulus response, are the cellular components of the nucleus and the membrane, and play molecular roles in enzyme regulation. KEGG pathway enrichment analysis revealed that PTs regulate the PI3K/Akt pathway. Genetic alterations for each target gene were MTOR (3%), NOTCH1 (4%), TP53 (42%), MMP9 (4%), NFKB1 (3%), PIK3CA (32%), PTGS2 (15%), and RELA (5%). The Kaplan–Meier plot showed that patients with low mRNA expression levels of MTOR, TP53, MMP9, NFKB1, PTGS2, and RELA and high expression of PIK3CA had a significantly better prognosis than their counterparts. Further validation of gene expression by using GEPIA revealed that the mRNA expression of MMP9 was significantly higher in breast cancer tissues than in normal tissues, whereas the mRNA expression of PTGS2 showed the opposite. Analysis with ONCOMINE demonstrated that the mRNA expression levels of MMP9 and NFKB1 were significantly higher in metastatic breast cancer cells than in normal tissues. The results of molecular docking analyses revealed the advantage of tangeretin as an inhibitor of PIK3CA, MMP9, PTGS2, and IKK. Tangeretin inhibits metastasis in breast cancer cells by targeting TP53, PTGS2, MMP9, and PIK3CA and regulating the PI3K/Akt signaling pathway. Further investigation is needed to validate the results of this study.

Role of Metastasis Suppressor KAI1/CD82 in Different Cancers.

Metastasis is one of the characteristics of malignant tumors and the main cause of death worldwide. The process of metastasis is mainly affected by tumor metastasis genes, tumor metastasis suppressor genes, tumor microenvironment, extracellular matrix degradation, and other factors. Thus, it is essential to elucidate the mechanism of metastasis and find the therapeutic targets in order to prevent the development of malignant tumors. KAI1/CD82, a member of tetraspanin superfamily of glycoproteins, has been reported as a tumor metastasis suppressor gene in various types of cancers without affecting the tumor formation. Many studies have demonstrated that low expression of KAI1/CD82 might lead to poor prognosis due to its interactions with other tetraspanins and integrins, resulting in the regulation of cell motility and invasion, cell-cell adhesion, and apoptosis. Considering its pathological and physiological significance, KAI1/CD82 could be a potential strategy for clinical predicting and preventing tumor progression and metastasis. The present review aims to discuss the role of KAI1/CD82 in metastasis for different cancers and examine its prospects as a metastasis biomarker and a therapeutic target.

Low expression of miR-29a is associated with aggressive biology and worse survival in gastric cancer

Advanced gastric cancer (GC) is one of the most lethal cancer types, thus a better understanding of its biology in patients is urgently needed. MicroRNA (miR)-29a is a known tumor suppressive miR that is related to metastasis, but its clinical relevance in GC remains ambiguous. Here, using a large GC patient cohort we hypothesized that low expression of miR-29a in GC is associated with aggressive cancer biology and worse survival. We demonstrated that low miR-29a GC enriched cell proliferation, apoptosis, metastasis, and angiogenesis related gene sets, as well as the higher expression of related genes. Low miR-29a GC was associated with less anti-cancer immune cell infiltration as well as immune related scoring. Low miR-29a GC demonstrated a worse overall survival (OS) as well as disease specific survival (DSS) compared with high expressing miR-29a GC. Notably, low miR-29a expression was the only factor, other than residual tumor status, to be an independent prognostic biomarker of worse OS and DSS. In conclusion, low miR-29a GC was associated with aggressive cancer biology and worse OS as well as DSS. Additionally, low expression of miR-29a was an independent prognostic biomarker of OS and DSS in gastric cancer patients.

Identification of the miRNA signature and key genes in colorectal cancer lymph node metastasis

BackgroundBecause its metastasis to the lymph nodes are closely related to poor prognosis, miRNAs and mRNAs can serve as biomarkers for the diagnosis, prognosis, and therapy of colorectal cancer (CRC). This study aimed to identify novel gene signatures in the lymph node metastasis of CRC.MethodsGSE56350, GSE70574, and GSE95109 datasets were downloaded from the Gene Expression Omnibus (GEO) database, while data from 569 colorectal cancer cases were also downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed miRNAs (DE-miRNAs) were calculated using R programming language (Version 3.6.3), while gene ontology and enrichment analysis of target mRNAs were performed using FunRich (http://www.funrich.org). Furthermore, the mRNA–miRNA network was constructed using Cytoscape software (Version 3.8.0). Gene expression levels were verified using the GEO datasets. Similarly, quantitative real-time PCR (qPCR) was used to examine expression profiles from 20 paired non-metastatic and metastatic lymph node tissue samples obtained from patients with CRC.ResultsIn total, five DE-miRNAs were selected, and 34 mRNAs were identified after filtering the results. Moreover, two key miRNAs (hsa-miR-99a, hsa-miR-100) and one gene (heparan sulfate-glucosamine 3-sulfotransferase 2 [HS3ST2]) were identified. The GEO datasets analysis and qPCR results showed that the expression of key miRNA and genes were consistent with that obtained from the bioinformatic analysis. A novel miRNA–mRNA network capable of predicting the prognosis and confirmed experimentally, hsa-miR-99a-HS3ST2-hsa-miR-100, was found after expression analysis in metastasized lymph node tissue from CRC samples.ConclusionIn summary, miRNAs and genes with potential as biomarkers were found and a novel miRNA–mRNA network was established for CRC lymph node metastasis by systematic bioinformatic analysis and experimental validation. This network may be used as a potential biomarker in the development of lymph node metastatic CRC.

MiR-622 acts as a tumor suppressor to induce cell apoptosis and inhibit metastasis in human prostate cancer.

Growing evidence indicating the critical modulator roles of microRNAs (miRNAs) involved in prostate cancer (PCa) metastasis that holds great promise as therapeutic targets. Herein, we transfected the miR-622 mimic into PC3 cells and evaluated the effects of this interference on these tumour cells' growth and the expression of specific metastatic genes. Transfecting of miR-622 mimic and inhibitor, negative control (NC) inhibitor and NC was established using Lipofectamine 2000. The mRNA levels of miR-622 and metastatic genes were evaluated using the qRT-PCR and Western blot. Cytotoxic effects of miR-622 were assessed by MTT. Apoptosis was detected using an ELISA cell death assay kit. miR-622 is down-regulated in PC3 cells. As expected, cell viability effects after transfection were described as miR-622 inhibitor >NC and NC inhibitor >miR-622 mimic (p<.01). Importantly, we showed that transfected miR-622 mimic could enhance the apoptosis of PC3 cells, while transfected miR-622 inhibitor could decrease cell apoptosis (p<.01). Furthermore, miR-622 overexpression could increase significantly down-regulated the MMP2, MMP9, CXCR-4, c-Myc and K-Ras expression levels. Findings demonstrate a novel mechanism by which miR-622 modulates PCa cells' metastasis by targeting metastatic genes. These data confirm the tumour-suppressive function of miR-622 in PCa cells by enhancing apoptosis and reducing metastasis.

Abstract 2310: Identification of novel essential genes for prostate cancer metastasis by genome scale CRISPR approaches

Abstract Background: Thanks to the availability of new biomarkers, Prostate cancer (PCa) can now be diagnosed earlier and, as a result, PCa patients have a high 5-year survival rate. Nonetheless, the situation changes completely for patients who develop metastasis, for whom 5-year survival rate decreases to 29%. Recent candidate-gene approaches have identified new candidates, but there are still no drugs to prevent or cure metastatic PCa. Hence, there is an urgent need to find effective therapeutic targets. Methodology: In this study we have conducted a high-throughput CRISPR/Cas9 screening in highly metastatic PCa cell lines PC3 and DU145, using the CRISPR/Cas9 knock-out (GeCKO) library, that targets 18,080 genes with 64,751 unique gRNAs. To assess whether each gene loss-of-function may be critical for metastasis development, invasion assays in Matrigel-coated Boyden Chamber membranes were used. Next-generation sequencing and bioinformatic MaGECK analysis were then carried out to identify novel key genes in the development of PCa metastasis. siRNA approach was used to further validate the best candidates. Results: We found 29 candidates and several signaling pathways capable of significatively impair the invasion of, both, PC3 and DU145 cell lines when knocked-out, being PRMT7 the strongest regulator. PRMT7 gene encodes a type II methyltransferase involved in several cellular processes such as mRNA splicing or DNA repair. There are evidences that indicate it regulates EMT through methylation of E-cadherin promoter and other targets. Moreover, its overexpression correlates with higher levels of expression of MMP9 and MMP2 that lead to metastasis appearance in other types of tumors. Our results inhibiting PRMT7 expression with siRNA validated our high-throughput CRISPR/Cas9 screening results and confirmed the implication of PRMT7 in PCa invasion. Conclusions: We have conducted a novel high-throughput CRISPR-Cas9 screening, finding promising gene candidates, such as PRMT7, which, in the future could be used as therapeutical targets to prevent metastasis development in PCa patients. Furthermore, in this study we have also found genes that had been previously associated to PCa metastasis in other candidate-gene studies, giving a proof-of-concept for the use of high performance of CRISPR-Cas9 screenings for the search of candidate genes involved in PCa metastasis. Citation Format: Maria Rodrigo Faus, Sara Manzano Figueroa, Jingchun Qu, Huiqi Qu, Renata Pellegrino, Hakon Hakonarson, Paloma Bragado, Almudena Porras, Alvaro Gutierrez-Uzquiza. Identification of novel essential genes for prostate cancer metastasis by genome scale CRISPR approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2310.

Abstract 2877: LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC)

Abstract Introduction: TNBC is associated with poorer prognosis compared to other types of breast cancer due to poorly understood aggressive metastatic behavior. Proteomic profiling prioritized Leucine-Zipper Protein 1 (LUZP1) as an understudied TNBC-specific protein from an analysis of breast cancer patient-derived xenografts (PDXs) [PMC5379071]. Approach: RNA-seq data and isobaric tags for relative and absolute quantitation (iTRAQ) data from 16 PDXs were used to identify differentially expressed genes in basal-like and claudin-low breast cancers, compared to luminal and HER2-enriched subtypes. Multiple datasets (DepMap, METABRIC, and TCGA) were used to validate the enrichment of LUZP1 in TNBC. T-test was applied to identify differential genes/proteins. Pearson correlation analysis was used to identify RNAs that are correlated with LUZP1 protein level in CTPAC proteogenomic clinical sample data [PMC5102256 and Cell in press]. Gene Set Enrichment Analysis (GSEA) was used to identify enriched pathways. Cell fractionation was used to determine the subcellular expression of LUZP1. qPCR was used to validate differential expression observed in RNA-seq. Results: Leucine-Zipper Protein 1 (LUZP1) was differentially elevated in basal-like and claudin-low PDX models versus luminal and HER2-enriched at the mRNA and protein levels across 16 PDXs. Furthermore, LUZP1 mRNA was significantly enriched in TNBCs, relative to luminal and HER2-enriched subtypes, in breast cancer cell lines (data from DepMap), and human breast tumors (data from METABRIC and TCGA). LUZP1 has three putative nuclear localization signals and three leucine-zipper motifs, thought to mediate its protein dimerization and DNA-binding properties. Fractionation of TNBC cells showed that LUZP1 was predominately present in the nucleus. RNA-sequencing of control and MDA-MB-231 LUZP1 knockdown cells revealed that several extracellular matrix (ECM)-related pathways and epithelial-to-mesenchymal transition (EMT) were regulated by LUZP1. In particular, expression of migration, invasion, and metastasis genes such as matrix metalloproteinases (MMPs), Bone Morphogenetic Protein 2 (BMP2), and Transforming Growth Factor Beta 2 (TGFB2) were found to be LUZP1 regulated. Additionally, in CPTAC breast cancer proteogenomic data, LUZP1-correlated proteins and transcripts were enriched in ECM-related pathways and EMT, confirming the pathophysiological relevance of the cell-line knock-down data. Conclusion: LUZP1 is a novel TNBC-enriched protein in breast cancer PDXs, cell lines, and human tumors data. Gene silencing experiments demonstrated that LUZP1 transcriptionally regulates metastasis-related genes, and thus may drive a transcriptional program responsible for invasion, metastasis and EMT in TNBC. Functional data will be presented. Citation Format: Junkai Wang, Doug Chan, Beom-Jun Kim, Gloria V. Echeverria, Eric Jaehnig, Bing Zhang, Matthew J. Ellis. LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2877.

Abstract 2851: Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT

Abstract Trop-2 is a type I transmembrane glycoprotein, which is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation. In its mature, glycosylated/functionally-competent form, Trop-2 associates with worse prognosis. A global quest was conducted for identifying decisive drivers of cancer metastasis. Cancer cell spheroids, wound healing and cell aggregation assays were utilized to assess cell-cell adhesion capacity. Immunofluorescence confocal microscopy and flow cytometry analysis were utilized to quantify expression of target signaling proteins, IHC analysis quantified the expression of target molecules in primary tumors and metastases. Pre-clinical models of orthotopic growth of colon cancer and metastatic diffusion to the liver were utilized. Xenotransplant transcriptome profiling analyzed transcription of EMT-related determinants. This led us to identify Trop-2 as a unique upregulated gene in colon cancer metastasis. Trop-2 was here shown to bind highly-expressed E-cadherin at the cell membrane. Trop-2 binding released E-cadherin from the cytoskeleton, inhibited cell-cell adhesion functions and activated signaling through β-catenin. This occurred in the absence of transcriptional downregulation of the CDH1 gene by EMT-associated transcription factors. The Trop-2/E-cadherin/β-catenin program was shown to lead to anti-apoptotic signaling, increased cell migration and enhanced cancer cell survival. In an orthotopic model of colon cancer, Trop-2 caused metastatic diffusion to the liver. The Trop-2-led E-cadherin-inactivation metastasis program was then found to induce metastatic relapse and shorter overall survival of colon cancer patients. Corresponding impact was indicated on breast, colon, ovary, uterus, stomach cancer metastatic diffusion, for broad impact on metastatic diffusion in human cancer. Acknowledgments:Support was provided by grants of Italian Ministry of Development (MI01_00424) and of University and Research (SCN_00558). M.T. was supported by the Programma Per Giovani Ricercatori “Rita Levi Montalcini” (Grant PGR12I7N1Z). Citation Format: Saverio Alberti, Emanuela Guerra, Valeria Relli, Rossano Lattanzio, Martina Ceci, Khouloud Boujnah, Valeria Garbo, Antonino Moschella, donato F. Altomare, Raffaella Depalo, Marco Trerotola. Trop-2 inactivates E-cadherin for metastatic diffusion in the absence of EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2851.

Abstract 174: In-depth transcriptomic comparisons provide novel insights into cancer metastasis

Abstract Comparison of the transcriptome between primary and metastatic cancer is frequently performed to study cancer metastasis; however, distilling plausible biology from the comparison results remains a big challenge. We show that in the differential expression (DE) analysis performed on bulk RNAseq data a large proportion of DE genes are identified due to technical issues instead of biological differences between primary and metastatic cancer cells. Next, we develop a computational pipeline DEBoost to minimize such artifacts and apply it to five cancer types. We perform comprehensive analysis on the retained tumor-intrinsic DE genes to explore the biological mechanisms underlying the metastasis of different cancer types. Many of the DE genes are associated with the progression of primary cancer, suggesting the differential expression may have already been established before metastasis. Clustering analysis on DE genes identifies CNVs that account for the transcriptomic change and we further confirm 19p13.12 amplification drives the metastasis of primary Basal-like breast cancer via activating NOTCH signaling pathway. We also find that cell cycling is more active in metastatic prostate cancer than in primary cancer, suggesting the difference of responses to treatment like cell cycling inhibitors. Finally, we show that some metastatic cancer cells tend to express genes whose expression is normally observed at the metastasis site and we experimentally validate this phenomenon in colorectal cancer liver metastasis. Our pan-cancer liver metastasis study provides novel biological insights into cancer metastasis and meanwhile demonstrates the feasibility of characterizing tumor-intrinsic DE genes in cancer metastasis using our computational pipeline. Citation Format: KE LIU, Benjamin S. Glicksberg, Shreya Paithankar, Bin Chen. In-depth transcriptomic comparisons provide novel insights into cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 174.