High-grade serous carcinoma has a poor prognosis, owing primarily to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the ...proteogenomics of ovarian cancer
, but a systematic examination of both the tumour and stromal compartments is critical in understanding ovarian cancer metastasis. Here we develop a label-free proteomic workflow to analyse as few as 5,000 formalin-fixed, paraffin-embedded cells microdissected from each compartment. The tumour proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several of the proteins that it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer-associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported ovarian cancer migration, proliferation and in vivo growth and metastasis. Expression of NNMT in CAFs led to depletion of S-adenosyl methionine and reduction in histone methylation associated with widespread gene expression changes in the tumour stroma. This work supports the use of ultra-low-input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.
The tumor microenvironment (TME) is a key determinant of metastatic efficiency. We performed a quantitative high-throughput screen (qHTS) of diverse medicinal chemistry tractable scaffolds (44,420 ...compounds) and pharmacologically active small molecules (386 compounds) using a layered organotypic, robust assay representing the ovarian cancer metastatic TME. This 3D model contains primary human mesothelial cells, fibroblasts, and extracellular matrix, to which fluorescently labeled ovarian cancer cells are added. Initially, 100 compounds inhibiting ovarian cancer adhesion/invasion to the 3D model in a dose-dependent manner were identified. Of those, eight compounds were confirmed active in five high-grade serous ovarian cancer cell lines and were further validated in secondary
and
biological assays. Two tyrosine kinase inhibitors, PP-121 and milciclib, and a previously unreported compound, NCGC00117362, were selected because they had potency at 1 μmol/L
Specifically, NCGC00117362 and PP-121 inhibited ovarian cancer adhesion, invasion, and proliferation, whereas milciclib inhibited ovarian cancer invasion and proliferation. Using
kinase profiling and immunoblotting, we found that milciclib targeted Cdk2 and Cdk6, and PP-121 targeted mTOR.
, all three compounds prevented ovarian cancer adhesion/invasion and metastasis, prolonged survival, and reduced omental tumor growth in an intervention study. To evaluate the clinical potential of NCGC00117362, structure-activity relationship studies were performed. Four close analogues of NCGC00117362 efficiently inhibited cancer aggressiveness
and metastasis
Collectively, these data show that a complex 3D culture of the TME is effective in qHTS. The three compounds identified have promise as therapeutics for prevention and treatment of ovarian cancer metastasis.
Emerging evidence has indicated that high-grade serous ovarian cancer (HGSOC) originates in the fallopian tube, where the earliest known genetic lesion is the mutation of TP53. In addition to such ...genetic changes, HGSOC is characterized by altered metabolism, including the production of oncogenic lipids such as lysophosphatidic acid (LPA). To understand the crosstalk between TP53 mutations and LPA signaling, we utilized primary fallopian tube epithelial cells (FTEC) engineered to overexpress mutant p53. We found that gain-of-function (GOF) p53 mutations downregulated the LPA-degrading enzyme lysophosphatidic acid phosphatase type 6 (ACP6), leading to upregulation of focal adhesion signaling in an LPA-dependent manner. Although highly expressed in normal fallopian tube epithelium, ACP6 expression was significantly reduced in ovarian cancer tumors and early in situ lesions. Downregulation of ACP6 in ovarian cancer cells was necessary and sufficient to support HGSOC proliferation, adhesion, migration, and invasion. Using mouse models of metastasis, we established that attenuation of ACP6 expression was associated with increased tumor burden. Conversely, overexpression of ACP6 suppressed invasive behavior. These data identify an involvement of oncogenic p53 mutations in LPA signaling and HGSOC progression through regulation of ACP6 expression.
Abstract
Mammalian target of rapamycin (mTOR) is a strong driver of tumorigenesis in multiple types of tumors, however targeting mTOR for cancer therapy has yielded only limited success. This may be ...explained in part by signaling redundancy with other pathways, such as CDK9 mTOR-Like (CTORC) complexes, recently described by our group. One of the binders common to mTORC1 and CTORC2 is La related protein 1 (LARP1). LARP1 influences ribosome biogenesis by regulating translation of 5'terminal oligopyrimidine tract (5’ TOP) containing mRNAs, which often encode ribosomal proteins and translation factors. Here, we validate LARP1 as common target of mTORC1 and CTORC2 as well as investigate its influence on leukemogenesis. To explore LARP1’s role in leukemogenesis we utilized CRISPR/CAS9 technology to create OCI-AML5 and U937 LARP1 knockout (KO) cells. Loss of LARP1 expression significantly diminished proliferation potential of AML cell lines both in vitro as well as in vivo when implanted into the flank of athymic nude mice as xenografts. Additionally, primary CD34+ leukemia cells were transfected with LARP1 targeting or scrambled siRNA and leukemic progenitor growth was assessed in methocellulose colony formation assays. We observed suppression of leukemic progenitors colony forming ability in LARP1 siRNA transfected cells, compared to the Ctrl siRNA transfected cells. We also determined enhanced sensitivity of Larp1 KO cell lines to standard chemotherapy agents such Azacitidine, Cytarabine and Venetoclax. We employed polysome profiling to compare global translation levels between Larp1 KO and control U937 cells. Our polysomal profiling results show that KO of LARP1 in U937 cells reduces global translation and ribosome biogenesis as expected as we observed a suppression of both monosomal and polysomal peaks. In summary, Larp1 is important for leukemogenesis and influences their response to treatment with currently used chemotherapy agents. These observations together suggest that LARP1 is a promising target for development of novel antileukemia approaches.
Citation Format: Dominik A Nahotko, Aneta H Baran, Mariafausta Fischietti, Elspeth M Beauchamp, Leonidas C Platanias. Role of LARP1 in the leukemogenesis of Acute Myeloid Leukemia abstract. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A39.
High grade serous ovarian cancer (HGSOC) is characterized by early metastasis throughout the peritoneal cavity and a poor clinical outcome. During the metastatic process, ovarian cancer cells ...interact with diverse cell populations, including mesothelial cells, adipocytes, and cancer‐associated fibroblasts (CAFs). Ovarian cancer CAFs have well‐described roles in promoting the growth and progression of ovarian cancer by secreting pro‐proliferative cytokines and oncogenic extracellular matrix components (ECM). To better understand the proteomic changes that occur during metastasis in both tumor and stromal compartments, laser‐capture microdissection was coupled to label‐free, quantitative proteomics to independently characterize the proteomes of both tumor and stromal compartments in a cohort of HGSOC patients. We identified a conserved stromal proteomic signature associated with metastasis which included high expression of nicotinamide N‐methyltransferase (NNMT) in the metastatic stroma. NNMT is a metabolic enzyme that catalyzes the methylation of nicotinamide and consumes S‐adenosyl methionine (SAM), thus depleting cellular methylation potential and reducing methylation of DNA and proteins. NNMT regulated the expression of thousands of genes and was necessary and sufficient for the reprogramming of normal fibroblasts to CAFs. Stromal NNMT expression altered multiple metabolic pathways and led to a significant reduction in cellular SAM pools. Attenuated DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) were associated with global changes in gene expression. Using genetic and small‐molecule inhibitor strategies, we found that stromal NNMT activity promoted the progression and metastasis of ovarian cancer. This study provides support to the idea that stromal methyltransferase activities can be targeted to normalize the metastatic stroma and should be further explored as treatment targets for cancer.
Support or Funding Information
This work was supported by a Marsha Rivkin Foundation award (MAE), National Cancer Institute (NCI) grants CA111882 and CA211916 (EL), the Harris Family Foundation (MAE and SDY), the Ludwig Institute for Cancer Research (EL), NIH grant CA175399 and DP2GM128199 (REM), V Foundation for Cancer Research (V2016‐020 to REM), the Körber Foundation/Körber European Science Prize (MM), the Max‐Planck Society for the Advancement of Science (MM), the Novo Nordisk Foundation (Grant agreement NNF14CC0001 and NNF15CC0001; FC and MM), and University of Chicago Cancer Center Support Grant P30CA014599.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Caspase malfunction in stem cells often precedes the appearance and progression of multiple types of cancer, including human colorectal cancer. However, the caspase‐dependent regulation of intestinal ...stem cell properties remains poorly understood. Here, we demonstrate that Dronc, the Drosophila ortholog of caspase‐9/2 in mammals, limits the number of intestinal progenitor cells and their entry into the enterocyte differentiation programme. Strikingly, these unexpected roles for Dronc are non‐apoptotic and have been uncovered under experimental conditions without epithelial replenishment. Supporting the non‐apoptotic nature of these functions, we show that they require the enzymatic activity of Dronc, but are largely independent of the apoptotic pathway. Alternatively, our genetic and functional data suggest that they are linked to the caspase‐mediated regulation of Notch signalling. Our findings provide novel insights into the non‐apoptotic, caspase‐dependent modulation of stem cell properties that could improve our understanding of the origin of intestinal malignancies.
Synopsis
Non‐apoptotic activation of the initiator caspase Dronc contributes to preserving intestinal progenitor cells in a quiescent, non‐proliferative and undifferentiated state under experimental conditions without epithelial replenishment demand.
The prevalent accumulation and transient activation of Dronc maintains intestinal progenitor cells in quiescence under non‐regenerative conditions.
Dronc promotes quiescence independent of the apoptosis program and in part through the negative and Dronc‐dependent regulation of Notch‐signalling.
Caspase activation can prevent gut hyperplasia, and act as a tumour‐suppressor factor through non‐apoptotic mechanisms related to the regulation of stem cells properties.
Non‐apoptotic activation of the initiator caspase Dronc contributes to preserving intestinal progenitor cells in a quiescent, non‐proliferative and undifferentiated state under experimental conditions without epithelial replenishment demand.
Platinum (Pt)‐based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt‐R). This work shows that Pt‐R OC cells increase intracellular ...cholesterol through uptake via the HDL receptor, scavenger receptor type B‐1 (SR‐B1). SR‐B1 blockade using synthetic cholesterol‐poor HDL‐like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR‐B1 and re‐sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR‐B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR‐B1 expression through SREBF2. Targeting SR‐B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt‐R OC.
Platinum resistant (Pt‐R) ovarian cancer (OC) cells increase intracellular cholesterol through uptake via the high‐density lipoproteins (HDL) receptor, scavenger receptor type B‐1 (SR‐B1). SR‐B1 blockade using HDL nanoparticles (NPs) diminishes cholesterol uptake and reduces GPx4 expression leading to Pt‐R OC cell death through ferroptosis, which represents a first‐in‐class approach applicable to Pt‐R OC.
Abstract
The cancer cells, stromal cells and stroma in the tumor microenvironment contribute to cancer metastasis and drug resistance. Therefore we applied an organotypic, robust and reliable assay ...for quantitative high throughput screening (qHTS) in drug discovery. This 1536-well HTS culture contains primary human mesothelial cells, fibroblasts, fibronectin and collagen type I and reproduces the human ovarian cancer (OvCa) metastatic microenvironment. The qHTS assay identifies small molecule compounds that inhibit OvCa adhesion/migration/invasion to the organotypic metastatic microenvironment.
Over 44,000 small molecules were screened in the primary qHTS assay. A counter screen was performed to eliminate any compounds toxic to the mesothelial cells or fibroblasts within the metastatic microenvironment. The activity of hit compounds was confirmed using five OvCa cell lines, and validated in secondary in vitro and in vivo biological assays. The active compounds directly inhibit at least two of three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion/invasion and metastasis, and improve survival in mouse models.
Collectively, these data indicate that the three compounds, two tyrosine kinase inhibitors and one novel compound, identified using an complex organtoypic culture of the tumor microenvironment for qHTS are potential therapeutics for OvCa metastasis.
Citation Format: Hilary A. Kenny, Madhu Lal, Min Shen, Dominik Nahotko, Sarah Fazal, Siquan Chen, Chun-Yi Chiang, Betul Kara, S. Diane Yamada, Kyle Brimacombe, Juan Marugan, Marc Ferrer and Ernst Lengyel. QUANTITATIVE HIGH-THROUGHPUT SCREENING USING AN ORGANOTYPIC MODEL IDENTIFIES TWO KINASE INHIBITORS THAT INHIBIT OVARIAN CANCER METASTASIS abstract. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-097.
PDF
