The significance of the phenotypic plasticity afforded by epithelial-mesenchymal transition (EMT) for cancer progression and drug resistance remains to be fully elucidated in the clinic. We evaluated ...epithelial-mesenchymal phenotypic characteristics across a range of tumor histologies using a validated, high-resolution digital microscopic immunofluorescence assay (IFA) that incorporates β-catenin detection and cellular morphology to delineate carcinoma cells from stromal fibroblasts and that quantitates the individual and colocalized expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (V) at subcellular resolution ("EMT-IFA"). We report the discovery of β-catenin
cancer cells that coexpress E-cadherin and vimentin in core-needle biopsies from patients with various advanced metastatic carcinomas, wherein these cells are transitioning between strongly epithelial and strongly mesenchymal-like phenotypes. Treatment of carcinoma models with anticancer drugs that differ in their mechanism of action (the tyrosine kinase inhibitor pazopanib in MKN45 gastric carcinoma xenografts and the combination of tubulin-targeting agent paclitaxel with the BCR-ABL inhibitor nilotinib in MDA-MB-468 breast cancer xenografts) caused changes in the tumor epithelial-mesenchymal character. Moreover, the appearance of partial EMT or mesenchymal-like carcinoma cells in MDA-MB-468 tumors treated with the paclitaxel-nilotinib combination resulted in upregulation of cancer stem cell (CSC) markers and susceptibility to FAK inhibitor. A metastatic prostate cancer patient treated with the PARP inhibitor talazoparib exhibited similar CSC marker upregulation. Therefore, the phenotypic plasticity conferred on carcinoma cells by EMT allows for rapid adaptation to cytotoxic or molecularly targeted therapy and could create a form of acquired drug resistance that is transient in nature. SIGNIFICANCE: Despite the role of EMT in metastasis and drug resistance, no standardized assessment of EMT phenotypic heterogeneity in human carcinomas exists; the EMT-IFA allows for clinical monitoring of tumor adaptation to therapy.
Sprouty (Spry) proteins are negative regulators of receptor tyrosine kinase signaling; however, their exact mechanism of action remains incompletely understood. We identified ...phosphatidylinositol-specific phospholipase C (PLC)-γ as a partner of the Spry1 and Spry2 proteins. Spry-PLCγ interaction was dependent on the Src homology 2 domain of PLCγ and a conserved N-terminal tyrosine residue in Spry1 and Spry2. Overexpression of Spry1 and Spry2 was associated with decreased PLCγ phosphorylation and decreased PLCγ activity as measured by production of inositol (1,4,5)-triphosphate (IP(3)) and diacylglycerol, whereas cells deficient for Spry1 or Spry1, -2, and -4 showed increased production of IP(3) at baseline and further increased in response to growth factor signals. Overexpression of Spry 1 or Spry2 or small-interfering RNA-mediated knockdown of PLCγ1 or PLCγ2 abrogated the activity of a calcium-dependent reporter gene, suggesting that Spry inhibited calcium-mediated signaling downstream of PLCγ. Furthermore, Spry overexpression in T-cells, which are highly dependent on PLCγ activity and calcium signaling, blocked T-cell receptor-mediated calcium release. Accordingly, cultured T-cells from Spry1 gene knockout mice showed increased proliferation in response to T-cell receptor stimulation. These data highlight an important action of Spry, which may allow these proteins to influence signaling through multiple receptors.
Recent studies have shown that activating mutations of NOTCH1 are responsible for the majority of T cell acute lymphoblastic leukemia (T-ALL) cases. Most of these mutations truncate its C-terminal ...domain, a region that is important for the NOTCH1 proteasome-mediated degradation. We report that the E3 ligase FBW7 targets NOTCH1 for ubiquitination and degradation. Our studies map in detail the amino acid degron sequence required for NOTCH1-FBW7 interaction. Furthermore, we identify inactivating FBW7 mutations in a large fraction of human T-ALL lines and primary leukemias. These mutations abrogate the binding of FBW7 not only to NOTCH1 but also to the two other characterized targets, c-Myc and cyclin E. The majority of the FBW7 mutations were present during relapse, and they were associated with NOTCH1 HD mutations. Interestingly, most of the T-ALL lines harboring FBW7 mutations were resistant to gamma-secretase inhibitor treatment and this resistance appeared to be related to the stabilization of the c-Myc protein. Our data suggest that FBW7 is a novel tumor suppressor in T cell leukemia, and implicate the loss of FBW7 function as a potential mechanism of drug resistance in T-ALL.
Notches, NF-kBs and the Making of T Cell Leukemia Aifantis, Iannis; Vilimas, Tomas; Buonamici, Silvia
Cell cycle (Georgetown, Tex.),
2/15/2007, 2007/02/15, 2007-02-15, Letnik:
6, Številka:
4
Journal Article
Recenzirano
Odprti dostop
T cell lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer frequent within pediatric ALL patients. Recent findings suggested that the transmembrane receptor NOTCH1 is the major ...oncogene for the majority of T-ALL cases. In these cases activating mutations of NOTCH1 are responsible for the transformation of developing T cell progenitors. These observations prompted us to study the mechanisms of Notch1-induced T cell transformation. Using parallel studies in T cell progenitors and established T-ALL lines we have demonstrated that the NF-kB signaling pathway is targeted and induced by Notch1 activation. Our studies suggested that the NF-kB activation by Notch1 can be direct, as Notch1 can bind and activate the promoters of the RELB and NFKB2 factors and indirect, as Notch1 can form a complex with the NF-kB kinase IKK. NF-kB appears to be important for the development of the disease as suppression of the pathway antagonizes T cell transformation both in vitro and in vivo, using animal models of T-ALL. We believe that these findings could be important for the understanding of Notch1 signaling and the therapeutic treatment of T-ALL.
Abstract
Background: A PDX bladder cancer model, BL0293-F563, grows large subcutaneous tumors, spontaneously metastasizes to the liver and bone, and sheds high numbers of circulating tumor cells ...(CTCs). This PDX model provides a unique opportunity to explore the relationships between primary tumors, CTCs and metastatic cell subpopulations. Methods: BL0293-F563 tumors (available from the NCI Patient-Derived Models Repository https://pdmr.cancer.gov/ and originally developed by Jackson Laboratories) were implanted into NSG mice and and primary tumors, metastatic nodules in the liver, and blood were collected at maximal allowable tumor burden. Tumor tissue was dissociated using Miltenyi Tumor Dissociation Kit with OctoDissociator, and Human CTCs were enriched from whole mouse blood through negative selection with anti-mouse CD45 and anti-mouse MHC-1 magnetic beads. Single cell sequencing was done using 10X Genomics 3’ gene expression assay v3.1. Sequencing libraries were prepared using 10X Genomics Chromium and 3’ gene expression kit v3.1. Data processing and analysis was done using 10X Genomics’ Cell Ranger pipeline, Seurat, and consensus non-negative matrix factorization. Results: Using Seurat FindNeighbors, cells in the aggregated dataset were classified into 17 distinct clusters. All clusters were comprised of cells from multiple sites (primary tumor, CTCs, metastases), but three clusters were enriched in CTCs and one cluster was composed of mostly primary tumor cells. All clusters exhibited an epithelial-like gene expression signature score, suggesting that CTC shedding was occurring without prominent epithelial-mesenchymal transition. Consistent with expected differences in oxygenation states, CTC-enriched clusters exhibited a lower hypoxia gene expression score than primary tumor and metastasis-enriched clusters. CTC-enriched clusters also showed higher expression of oxidative phosphorylation genes, suggesting metabolic differences between CTCs and cells from primary tumors and metastases. Based on Human Primary Cell Atlas phenotype prediction, several clusters were associated with stem cell like phenotypes. Additionally, two of three CTC-enriched clusters had elevated expression of mitosis-associated genes, suggesting that at least some populations of CTCs are not quiescent but actively cycling. Conclusions: Utilizing single cell gene expression profiling, we have linked the gene expression profile of CTCs to specific cell subpopulations in primary tumors and metastases. We show that CTC-enriched cell clusters appear to maintain an epithelial phenotype. Subpopulations of CTC cells exhibited enrichment of stemness-associated transcripts and features of active cell cycling.
Citation Format: Tomas Vilimas, Brandie Fullmer, Alyssa Chapman, Li Chen, Ting-Chia Chang, Rini Pauly, Biswajit Das, Chris Karlovich, Yvonne A. Evrard, Howard Stotler, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Melinda G. Hollingshead, James H. Doroshow, P. Mickey Williams. Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P097.
Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used ...the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.
Abstract
Background: A major set of preclinical models derived from specimens acquired from rapid autopsy patients in the National Cancer Institute Patient-Derived Models Repository (NCI PDMR, ...https://pdmr.cancer.gov) were from pancreatic adenocarcinoma (PAAD) patients, with metastatic specimens originating from liver, colon, omentum, and lung. Genomic characterization of these preclinical models provides a unique opportunity to study tumor heterogeneity and subclonality associated with the metastatic process and potential treatment resistance.
Methods: To date, 30 rapid autopsy patient-derived xenograft (PDX)/patient-derived cell (PDC)/patient-derived organoid (PDOrg ) models derived from pancreatic adenocarcinoma patients (n = 9) have been sequenced using whole-exome sequencing (WES) and RNASeq. Tumor heterogeneity between primary and metastatic sites was studied based on somatic mutation, copy number alteration (CNA) and gene expression data. A bioinformatics workflow was developed to stably infer and visualize the tumor subclonality by integrating the tools of PyClone, SCHISM, and TIMESCAPE, using somatic mutations and site-specific copy number data of multiple samples generated from PDX models in primary and metastatic sites.
Results: Among 30 rapid autopsy preclinical models from primary and metastatic sites, liver is the most common metastatic site in PAAD (9/19=47%) compared to other sites. Driver mutations are conserved in all preclinical model specimens derived from a given patient. KRAS p.G12D is present in 28 PDX/PDC/PDOrg models as well as the corresponding patient specimens, and BRAF p.V600E is present in other preclinical models. The fraction of the genome affected by CNA remains stable within a PDX model across passages (n=18, mean=7.63%, sd=5.90%). However, we found that this increased when comparing PDX models derived from metastatic sites versus the primary site (n=16, mean=19.47%, sd=9.69%). This indicates the presence of tumor heterogeneity between metastatic and primary sites. Site-specific subclones were identified in PDX models from two patients (521955 and 485368) and a phylogenetic tree of primary and metastasis sites indicates that one liver metastasis had a unique seeding event compared to the other metastatic sites for both patients.
Conclusion: Tumor heterogeneity and subclonality was observed in preclinical models generated from PAAD patients in the NCI PDMR. These models provide a unique resource for preclinical studies in tumor evolution, metastatic spread mediators, and drug resistance.
Citation Format: Li Chen, Biswajit Das, Yvonne A. Evrard, Chris A. Karlovich, Tomas Vilimas, Amanda Peach, Chapman Alyssa, Nikitha Nair, Anna L. Fong, Luis Romero, Ting-Chia Chang, Shahanawaz Jiwani, Lindsay Dutko, Kelly Benauer, Marianne Morton, Kelly Dougherty, Michelle A. Eugeni, Dianne Newton, Melinda G. Hollingshead, P. Mickey Williams, James H. Doroshow. Study of tumor heterogeneity and subclonality in primary pancreatic and metastatic sites from rapid autopsy patients in PDMR 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 3139.
Pre‐T‐cell receptor (pre‐TCR) functions and the study of early thymocyte development continue to fascinate immunologists more than 10 years after the first description and cloning of the receptor. ...Although multiple reports have addressed several aspects of pre‐TCR signaling and function, its ability to regulate diverse functions, including proliferation, survival, and allelic exclusion of the TCR‐β locus, remains an open question. What fascinates us is its central role in the fine balance between physiological differentiation and thymocyte transformation that leads to T‐cell leukemia and lymphomas. In this review, we integrate pre‐TCR signaling pathways and study their effects on the regulation of T‐cell progenitor cell‐cycle entry and cell survival. We also connect aberrant pre‐TCR signaling to deregulated proliferation and apoptotic balances and thymocyte transformation.
Abstract only
e13506
Background: The National Cancer Institute has developed a repository of preclinical models Patient-Derived Models Repository (NCI PDMR, https://pdmr.cancer.gov ) including ...patient derived xenografts (PDXs), organoids (PDOrgs) and in vitro tumor cultures (PDCs) from patients with solid tumor cancer histologies. A subset of these preclinical models is derived from post-mortem collections from rapid autopsies representing the end point in disease progression. Clinical annotations and genomic datasets associated with these models provide a unique opportunity to study tumor evolution, mechanistic insights into the metastatic process, and treatment resistance. Methods: To date, 43 PDXs, 21 PDCs, and 23 PDOrgs using rapid autopsy specimens from 8 primary and 35 metastatic sites of 18 patients have been developed by the Biological Testing Branch (DTP, DCTD, NCI Frederick, MD) for the PDMR. Whole exome (WES) and total transcriptome (RNASeq) data were processed to generate mutation, copy number alteration (CNA) and gene expression data. Multi-model lineage trees were reconstructed based on putative somatic variants for all the models derived from all patients. The fraction of the genome affected by CNA was compared both within and across PDX models. Results: Most of the rapid autopsy PDX models (32/43) are derived from pancreatic adenocarcinoma (PAAD) patients (13/18), with metastatic specimens originating from sites including liver, colon, omentum, and lung. Driver mutations are present in all preclinical model specimens derived from the same patient. For instance, KRAS p.G12D is present in all patient-derived model specimens derived from PAAD patient 521955. The fraction of the genome affected by CNA remains stable within a PDX model across passages (n = 24, mean = 6.39%, sd = 5.90%). However, we found that this increased when comparing PDX models derived from metastatic sites versus the primary site (n = 19, mean = 16.92%, sd = 10.46%). This indicates presence of tumor heterogeneity between metastatic and primary sites. The lineage tree for models from patient 521955 indicates that one liver metastasis has a unique seeding event compared to the other 4 metastatic sites. Unsupervised clustering analysis on gene expression data also confirms the observed tumor site relationships. Conclusions: Our data demonstrate the potential use of these preclinical models available from the NCI PDMR. These models provide a unique resource for preclinical studies in tumor evolution, metastatic spread mediators, and drug resistance.
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