CBIO-12. KAT5 ACTIVITY CONTROLS GLIOBLASTOMA CELL CYCLE DYNAMICS AND TUMOR GROWTH

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi29-vi29
Author(s):  
Anca Mihalas ◽  
Heather Feldman ◽  
Sonali Arora ◽  
John Bassett ◽  
Anoop Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Genetically Engineered ◽  
Lower Grade ◽  
Current Standard ◽  
Primary Tumors ◽  
A Genome ◽  
G0 Phase ◽  
Cell Cycle Dynamics

Abstract Current standard of care therapy for glioblastoma (GB) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how GB cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GB stem-like cells (GSCs) harboring a G0 reporter construct, which is stabilized when cells enter a G0-like state. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that knockout of KAT5 in vitro and in vivo dramatically increases G0 subpopulations in GSC cultures and GSC-induced tumors. Using genetically engineered GSC harboring KAT5 under the control of a Doxycyclin-titratable promoter, we establish that incrementally down regulating KAT5 activity is sufficient to slow cell cycle dynamics causing a build-up G0-like cells; and that partial inhibition of KAT5 leads to extended (mouse) patient survival. Further, in primary tumors, cell-based KAT5 activity assays revealed that high grade tumors harbor larger cell subpopulations with higher KAT5 activity than lower grade tumors. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors, may contribute to tumor progression, and may provide novel therapeutic opportunities.

scholarly journals CBMT-42. A GENOME-WIDE CRISPR-Cas9 SCREEN FOR GENES REGULATING QUIESCENT-LIKE STATES IN GLIOBLASTOMA

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi42-vi42
Author(s):  
Heather Feldman ◽  
Anca Mihalas ◽  
Christopher Plaisier ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Tumor Cells ◽  
Standard Of Care ◽  
Cell Types ◽  
Phenotypic Characterization ◽  
Current Standard ◽  
Genome Wide ◽  
A Genome ◽  
G0 Phase

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, non-cycling, quiescent-like states (G0 phase cells) are present in both normal tissue and tumors and play important roles in maintaining heterogeneity and cellular hierarchies. The presence of quiescent-like/G0 states therefore represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. However, it remains largely unclear to what degree or by what mechanisms tumor cells enter into or exit from quiescent-like states. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a p27-mVenus reporter construct, which is stabilized when cells enter a G0-like state. By assaying p27 reporteractivity, we were able to identify sgRNAs enriched in p27hipopulations and, which upon retest, trigger a G0-like arrest in GSCs. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, including KAT5 itself. Remarkably, we show that downregulation of KAT5 in vitro and in vivo dramatically increases the pool of cells in G0-like states in GSC cultures and GSC-induced tumors. Using single cell RNA-sequencing, we show that this cell state is characterized by gene expression signatures similar to those found in non-dividing subpopulations of GBM tumors and quiescent neural stem cells. In addition, we perform in-depth molecular and phenotypic characterization of these induced G0-like states, including epigenetic and metabolic profiles. These suggest a key role for KAT5 in regulating genes related to protein synthesis. In summary, our results suggest that Tip60/KAT5 activity plays key roles in G0 ingress/egress for GBM tumors and may provide novel therapeutic opportunities.

scholarly journals An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma

2020 ◽  
Author(s):  
Rediet Zewdu ◽  
Elnaz Mirzaei Mehrabad ◽  
Kelley Ingram ◽  
Alex Jones ◽  
Soledad A. Camolotto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Lung Tumor ◽  
Genetically Engineered ◽  
Reciprocal Relationship ◽  
Response To Therapy ◽  
Response To Treatment ◽  
Mek Inhibitors

ABSTRACTCancer cells often undergo lineage switching during their natural progression and in response to therapy. Lung adenocarcinomas (LUADs) exhibit a variety of differentiation states accompanied by dysregulation of lineage-specific transcription factors such as NKX2-1. Loss of NKX2-1 in human and murine LUAD leads to invasive mucinous adenocarcinoma (IMA), a subtype of lung cancer that exhibits pulmonary to gastric transdifferentiation. Human IMAs harbor a distinct spectrum of mutationally activated driver oncogenes compared to LUAD overall, suggesting that the transdifferentiation induced by NKX2-1 loss plays a context-dependent role in LUAD progression. Using genetically engineered mouse models, we find that NKX2-1 is required for optimal BRAFV600E driven lung tumor initiation but is dispensable for growth of established lung tumors. NKX2-1-deficient, BRAFV600E driven tumors morphologically resemble human IMA, have high levels of ERK phosphorylation and exhibit a distinct response to treatment with combined BRAF/MEK inhibitors. Whereas NKX2-1-positive tumor cells enter quiescence when treated with BRAF/MEK inhibitors, residual NKX2-1-negative cells fail to exit the cell cycle in response to the same therapy. Additionally, BRAF/MEK inhibitors induce canonical WNT signaling in NKX2-1-negative lung tumor cells, which is accompanied by cell identity switching within the gastric lineage. Co-inhibition of MAPK and WNT pathways blocked elements of this lineage switch in vitro and interfered with cell cycle changes imposed by MAPK inhibition in vivo. Our data show that there is a complex and reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of LUAD identity and suggest that lineage switching induced by targeted therapies may confer new therapeutic vulnerabilities.

In vitro chemotherapy profiling of well-differentiated midgut neuroendocrine tumors (NETs) based on individual patient tumor biomarkers analysis.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 235-235
Author(s):  
Yi-Zarn Wang ◽  
Jean P. Carrasquillo ◽  
Alexis Carimi ◽  
Elizabeth McCord ◽  
Maria M. Chester ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Neuroendocrine Tumors ◽  
Tumor Biomarker ◽  
Primary Tumors ◽  
Sensitivity Assessment ◽  
Biomarker Analysis ◽  
Well Differentiated

235 Background: Midgut neuroendocrine tumors (NETs) are rare malignancies with indolent clinical courses. In general, they are well differentiated with most tumor cells in the G0 phase of the cell cycle, consistent with the poor response rate of NETs to chemotherapy in vivo. We hypothesize that insults, such as surgery, can drive NET cells from G0 into S phase and that biomarker analysis of individual patient tumors harvested and grown in the lab will provide useful practical guide for future intra and post operative adjuvant therapy. Methods: 97 well-differentiated midgut NET patients underwent cytoreductive surgery at our institution between 5/2012 and 10/2012. 148 surgical specimens were collected and submitted to a single commercial lab for processing. Primary tumors, lymph nodes and liver metastases were harvested and cultured. Their RNAs were then extracted to analyze the expressivity a total of 88 different biomarkers. Based on our patients specific tumor biomarker expressivity and known correlations between 36 anti-neoplastic agents with their linked biomarkers, recommendations were reported as clinically benefit or lacking such benefit. Results: A total of 148 specimens from 97 patients were tested. In four of the 97 patients, no clinically beneficial chemotherapy agent could be identified. Among the remaining 93 patients, the top three agents that are most likely to be clinically beneficial are: Fluorouracil, Cisplatin and Carboplatin. These were reported to be clinically beneficial in 135/148 (91.2%), 103/148 (69.6%), and 103/148 (69.6%) patients respectively. Conclusions: Midgut NETs are slow growing tumors which are chemotherapeutically inert owing to the fact that most of the tumor cells are in G0 cell cycle. Surgical insult drives NET cells into active synthetic phase where they begin to express biomarkers specific to their tumor cells. Analysis of these biomarkers guides further potential beneficial therapy based on the current known associations among biomarkers and chemotherapy agents. These results must then be compared and confirmed against a direct in-vitro chemo sensitivity assessment conducted simultaneously on the same patients.

scholarly journals CBIO-19. LOW GLOBAL HISTONE H4 ACETYLATION LEVELS REVEAL CANDIDATE QUIESCENT CELL POPULATIONS IN GLIOMA AND DISTINGUISH TUMORS BY GRADE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii19-ii19
Author(s):  
Anca Mihalas ◽  
Heather Feldman ◽  
Anoop Patel ◽  
Patrick Paddison
Keyword(s):  
Cell Types ◽  
Strand Break ◽  
Cell Cycle Gene ◽  
Low Grade ◽  
Histone H4 ◽  
Current Standard ◽  
A Genome ◽  
Histone H4 Acetylation

Abstract Current standard of care therapy for glioblastoma (GBM) includes cytoreduction followed by ablative therapies that target rapidly dividing cell types. However, the presence of quiescent-like/G0 states, therefore, represents a natural reservoir of tumor cells that are resistant to current treatments. Quiescence or G0 phase is a reversible state of “stasis” cells enter in response to developmental or environmental cues. To gain insight into how glioblastoma cells might regulate G0-like states, we performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) harboring a G0-reporter to identify genes that when inhibited trap GSCs in G0-like states. Among the top screen hits were members of the Tip60/KAT5 histone acetyltransferase complex, which targets both histones (e.g., H4) and non-histone proteins for acetylation. NuA4 functions as a transcriptional activator, whose activities are coordinated with MYC in certain contexts, and also participates in DNA double-strand break repair by facilitating chromatin opening. However, currently little is known about the roles for NuA4 complex in GBM biology. Through modeling KAT5 function in GSC in vitro cultures and in vivo tumors, we find that KAT5 inhibition causes cells to arrest in a G0-like state with high p27 levels, G1-phase DNA content, low protein synthesis rates, low rRNA rates, lower metabolic rate, suppression of cell cycle gene expression, and low histone H4 acetylation. Interestingly, partial inhibition of KAT5 activity slows highly aggressive tumor growth, while increasing p27hi H4-aclow populations. Remarkably, we that low grade gliomas have significantly higher H4-aclow subpopulations and generally lower H4-ac levels than aggressive grade IV tumors. Taken together, our results suggest that NuA4/KAT5 activity may play a key role in quiescence ingress/egress in glioma and that targeting its activity in high grade tumors may effectively “down grade” them, thus, increase patient survival.

scholarly journals Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression.

1995 ◽  
Vol 15 (1) ◽  
pp. 552-560 ◽  
Author(s):  
M Hattori ◽  
N Tsukamoto ◽  
M S Nur-e-Kamal ◽  
B Rubinfeld ◽  
K Iwai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Nuclear Protein ◽  
Interleukin 2 ◽  
Quiescent State ◽  
Cycle Progression ◽  
Ran Gtpase ◽  
G0 Phase ◽  
Nih 3T3

We have cloned a novel cDNA (Spa-1) which is little expressed in the quiescent state but induced in the interleukin 2-stimulated cycling state of an interleukin 2-responsive murine lymphoid cell line by differential hybridization. Spa-1 mRNA (3.5 kb) was induced in normal lymphocytes following various types of mitogenic stimulation. In normal organs it is preferentially expressed in both fetal and adult lymphohematopoietic tissues. A Spa-1-encoded protein of 68 kDa is localized mostly in the nucleus. Its N-terminal domain is highly homologous to a human Rap1 GTPase-activating protein (GAP), and a fusion protein of this domain (SpanN) indeed exhibited GAP activity for Rap1/Rsr1 but not for Ras or Rho in vitro. Unlike the human Rap1 GAP, however, SpanN also exhibited GAP activity for Ran, so far the only known Ras-related GTPase in the nucleus. In the presence of serum, stable Spa-1 cDNA transfectants of NIH 3T3 cells (NIH/Spa-1) hardly overexpressed Spa-1 (p68), and they grew as normally as did the parental cells. When NIH/Spa-1 cells were serum starved to be arrested in the G1/G0 phase of the cell cycle, however, they, unlike the control cells, exhibited progressive Spa-1 p68 accumulation, and following the addition of serum they showed cell death resembling mitotic catastrophes of the S phase during cell cycle progression. The results indicate that the novel nuclear protein Spa-1, with a potentially active Ran GAP domain, severely hampers the mitogen-induced cell cycle progression when abnormally and/or prematurely expressed. Functions of the Spa-1 protein and its regulation are discussed in the context of its possible interaction with the Ran/RCC-1 system, which is involved in the coordinated nuclear functions, including cell division.

scholarly journals CRISPR/Cas9 Editing of the Polyomavirus Tumor Antigens Inhibits Merkel Cell Carcinoma Growth In Vitro

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1260 ◽  
Author(s):  
Arturo Temblador ◽  
Dimitrios Topalis ◽  
Graciela Andrei ◽  
Robert Snoeck
Keyword(s):  
Cell Cycle ◽  
Cell Carcinoma ◽  
Tumor Cells ◽  
Merkel Cell Carcinoma ◽  
Tumor Antigens ◽  
Therapeutic Option ◽  
Target Sequence ◽  
Merkel Cell ◽  
Guide Rna

Merkel cell carcinoma (MCC) is an aggressive type of skin cancer whose main causative agent is Merkel cell polyomavirus (MCPyV). MCPyV is integrated into the genome of the tumor cells in most MCCs. Virus-positive tumor cells constitutively express two viral oncoproteins that promote cell growth: the small (sT) and the large (LT) tumor antigens (TAs). Despite the success of immunotherapies in patients with MCC, not all individuals respond to these treatments. Therefore, new therapeutic options continue to be investigated. Herein, we used CRISPR/Cas9 to target the viral oncogenes in two virus-positive MCC cell lines: MS-1 and WAGA. Frameshift mutations introduced in the target sequence upon repair of the Cas9-induced DNA break resulted in decreased LT protein levels, which subsequently impaired cell proliferation, caused cell cycle arrest, and led to increased apoptosis. Importantly, a virus-negative non-MCC cell line (HEK293T) remained unaffected, as well as those cells expressing a non-targeting single-guide RNA (sgRNA). Thus, we presumed that the noted effects were not due to the off-target activity of the TAs-targeting sgRNAs. Additionally, WAGA cells had altered levels of cellular proteins involved in cell cycle regulation, supporting the observed cell cycle. Taken together, our findings provide evidence for the development of a CRISPR/Cas9-based therapeutic option for virus-positive MCC.

scholarly journals Tumor quiescence: elevating SOX2 in diverse tumor cell types downregulates a broad spectrum of the cell cycle machinery and inhibits tumor growth

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ethan P. Metz ◽  
Erin L. Wuebben ◽  
Phillip J. Wilder ◽  
Jesse L. Cox ◽  
Kaustubh Datta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Tumor Growth ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Cell Cycle Machinery

Abstract Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

scholarly journals GROWTH INHIBITION OF MURINE TUMOR CELLS, IN VITRO, BY PUROMYCIN, [6N]O2'-DIBUTYRYL 3',5'-ADENOSINE MONOPHOSPHATE, OR ADENOSINE

1973 ◽  
Vol 57 (2) ◽  
pp. 397-405 ◽  
Author(s):  
D. B. Thomas ◽  
Gay Medley ◽  
C. A. Lingwood
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Growth Arrest ◽  
Adenosine Monophosphate ◽  
Synchronous Cultures ◽  
Murine Tumor Cells

The cytostatic effects of puromycm, [6N]O2'-dibutyryl 3',5'-adenosine monophosphate, and adenosine on asynchronous and synchronous cultures of the murine mastocytoma, P815Y, have been studied. Cell growth was arrested after a minimum of one further division. A model is proposed for the inhibition of cell division in which the periods of inhibition and growth arrest are separated in time by one cell cycle.

scholarly journals SDF-1α/MicroRNA-134 Axis Regulates Nonfunctioning Pituitary Neuroendocrine Tumor Growth via Targeting VEGFA

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiaoyu Wang ◽  
Yuanjian Fang ◽  
Yunxiang Zhou ◽  
Xiaoming Guo ◽  
Ke Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Neuroendocrine Tumor ◽  
Tumor Cells ◽  
Tumor Invasion ◽  
Expression Level ◽  
Qrt Pcr ◽  
Proliferation And Invasion

BackgroundNonfunctioning pituitary neuroendocrine tumor (NF-PitNET) is difficult to resect. Except for surgery, there is no effective treatment for NF-PitNET. MicroRNA-134 (miR-134) has been reported to inhibit proliferation and invasion ability of tumor cells. Herein, the mechanism underlying the effect of miR-134 on alleviating NF-PitNET tumor cells growth is explored.MethodsMouse pituitary αT3-1 cells were transfected with miR-134 mimics and inhibitor, followed by treatment with stromal cell-derived factor-1α (SDF-1α) in vitro. MiR-134 expression level: we used quantitative real-time PCR (qRT-PCR) to detect the expression of miR-134. Cell behavior level: cell viability and invasion ability were assessed using a cell counting kit-8 (CCK8) assay and Transwell invasion assay respectively. Cytomolecular level: tumor cell proliferation was evaluated by Ki-67 staining; propidium iodide (PI) staining analyzed the effect of miR-134 on cell cycle arrest; western blot analysis and immunofluorescence staining evaluated tumor migration and invasive ability. Additionally, we collected 27 NF-PitNET tumor specimens and related clinical data. The specimens were subjected to qRT-PCR to obtain the relative miR-134 expression level of each specimen; linear regression analysis was used to analyze the miR-134 expression level in tumor specimens and the age of the NF-PitNET population, gender, tumor invasion, prognosis, and other indicators.ResultsIn vitro experiment, miR-134 was observed to significantly inhibit αT3-1 cells proliferation characterized by inhibited cell viability and expressions of vascular endothelial growth factor A (VEGFA) and cell cycle transition from G1 to S phase (P < 0.01). VEGFA was verified as a target of miR-134. Additionally, miR-134-induced inhibition of αT3-1 cell proliferation and invasion was attenuated by SDF-1α and VEGFA overexpression (P < 0.01). In primary NF-PitNET tumor analysis, miR-134 expression level was negatively correlated with tumor invasion (P = 0.003).ConclusionThe regulation of the SDF-1α/miR-134/VEGFA axis represents a novel mechanism in the pathogenesis of NF-PitNETs and may serve as a potential therapeutic target for the treatment of NF-PitNETs.

scholarly journals Rapid proliferation of activated lymph node CD4+ T cells is achieved by greatly curtailing the duration of gap phases in cell cycle progression

2014 ◽  
Vol 19 (4) ◽  
Author(s):  
Takuya Mishima ◽  
Shoko Toda ◽  
Yoshiaki Ando ◽  
Tsukasa Matsunaga ◽  
Manabu Inobe
Keyword(s):  
Cell Cycle ◽  
Immune Response ◽  
T Cells ◽  
Cd4 T Cells ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Cycle Times ◽  
Peripheral T Cells ◽  
G0 Phase

AbstractPeripheral T cells are in G0 phase and do not proliferate. When they encounter an antigen, they enter the cell cycle and proliferate in order to initiate an active immune response. Here, we have determined the first two cell cycle times of a leading population of CD4+ T cells stimulated by PMA plus ionomycin in vitro. The first cell cycle began around 10 h after stimulation and took approximately 16 h. Surprisingly, the second cell cycle was extremely rapid and required only 6 h. T cells might have a unique regulatory mechanism to compensate for the shortage of the gap phases in cell cycle progression. This unique feature might be a basis for a quick immune response against pathogens, as it maximizes the rate of proliferation.

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