Abstract
The presence of quiescent, therapy-resistant population of tumor cells is often attributed to extreme metastasis and tumor recurrence. This population, often described as cancer stem cells ...(CSCs), or tumor-initiating cells (TICs), is enriched in a tumor as a result of microenvironmental (hypoxia) or chemotherapeutic stress. In pancreatic cancer, CD133+ cells have been described as a representation of this aggressive TIC population and are most responsible for tumor relapse in patients. This population adapts to stress by turning on mechanisms to halt cell cycle progression. Upon removal of the stress, these cells restart their cell cycle and regain their proliferative nature. Growth Arrest Specific 5 (GAS5) is a long noncoding RNA critical for this process and is overexpressed in PDAC. 13C-Glucose labeling was used for glucose flux assay to track the use of glucose and other metabolites in CD133+ and CD133- populations of pancreatic cancer cells. CD133+/- cell populations were sorted with magnetic sorting or flow cytometry sorting techniques. Lentiviral stable transfection of human CD133 cDNA in MIA PaCa-2 cell line was used to obtain a CD133 overexpressing cell line. siGAS5 and siSOX2 were used to knock down genes in CD133Hi cell lines. Glucocorticoid receptor (GR) activity was assessed using GRE dual luciferase assay system (Qiagen). MTT-based assay was used for proliferation and BrDU incorporation assay was used for measure of nucleic acid synthesis. Fluorescent in situ hybridization using human GAS5 RNA probe (Stellaris) was used to visualize GAS5 localization in human PDAC vs. normal adjacent FFPE samples as well as xenograft FFPE samples. Our results show for the first time that the emergence of CD133+ population coincides with upregulation of GAS5, which reprograms cell cycle to slow proliferation by inhibiting GR-mediated cell cycle control. The CD133+ population further routed glucose through the pentose phosphate pathway, a predominantly biosynthetic pathway, in spite of being quiescent in nature. However, this did not result in immediate nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from growth arrest and restarted nucleic acid synthesis and proliferation. Additionally, this study shows that GAS5 regulates cell cycle through GR, which it is known to bind and inhibit. CD133Hi cells with high GAS5 expression had low GR activity, and stimulation of these cells with dexamethasone was able to increase cell proliferation. Our study thus shows that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, allowing these cells to overcome chemical and environmental stressors and leading to the aggressive nature of pancreatic tumors.
Citation Format: Nikita S. Sharma, Brittany C. Durden, Prisca Gnamlin, Vineet K. Gupta, Kousik Kesh, Vanessa T. Garrido, Ashok Saluja, Sulagna Banerjee. Long noncoding RNA growth arrest specific 5 (GAS5) as a proliferation «brake» in aggressive population of CD133+ cells responsible for recurrence in PDAC abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B17.
Abstract
Background: Pancreatic Cancer is an extremely aggressive disease with a bad outcome owing to its late detection, high rate of metastasis and self-renewal post-surgical and chemotherapeutic ...intervention. In cancer the balance between differentiation and self-renewal is tightly regulated by SOX2. Over expression of SOX2, as in pancreatic cancer, can tilt the balance towards self-renewal and thus increase the population of these undifferentiated “stem cells”.
Methods: A CRISPR based OGT knockout kit was purchased from Origene and a knockout cell line was generated with S2VP10 cells. For the tumor initiation studies OGTi (OGT knockout cells) were implanted subcutaneously at a limiting dilution from 500,000 cells to 500 cells. Site directed mutagenesis was performed using theQuick-change Lightning from Agilent.
Results: In the current study we show for the first time that SOX2 undergoes a type of post translational modification known as O GlcNAcylation. We also show for the first time that in humans this modification happens at Serine 246 and mutating this particular site via a side directed mutagenesis leads to a loss of O GlcNAcylation of SOX2.O GlcNAc modification of SOX2 further leads to its activation which then leads to its stabilization in the nucleus. A CRISPR-OGT knockout in pancreatic cancer cell line S2VP10 resulted in a delayed tumor initiation. We further showed that mutation of this site (S246A) prevents the modification of Sox2 and its downstream activity. Our study also demonstrated that targeting OGT in vivo with a small molecule inhibitor OSMI, results in decreased tumor burden, delayed tumor progression and a decreased expression of SOX2 in pancreatic cancer cells.
Conclusion: Our study highlights for the first time that the O-GlcNAc transferase dependent SOX2 glycosylation has a profound effect on the transcriptional activity of SOX2 and is instrumental in determining self-renewal in pancreatic cancer which can be targeted therapeutically.
Citation Format: Nikita S. Sharma, Vineet K. Gupta, Patricia Dauer, Roey Hadad, Kousik K. kesh, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. O GlcNAcylation of SOX2 regulates its tumor initiation properties in pancreatic cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4498.
Abstract
Background: Pancreatic adenocarcinoma (PDAC) is an extremely immunosuppressive and heterogenous neoplasm for which immune checkpoint inhibitor therapy has not been very successful. ...Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on one hand and also influence the extracellular matrix in the tumor microenvironment. Our study aims to sensitize pancreatic cancer to anti PD-1 therapy by targeting the HBP pathway.
Methods: 6-Diazo-5-oxo L-norleucine or DON was used as a GFAT inhibitor. Tetracycline-inducible GFAT knockout cell lines were made using S2-VP10 as the parent cell line.
Results: High-dose monotherapy of DON (1mg/kg/3days a week) caused significant decrease in tumor volume in immunocompetent KPC mice along with significant reduction in metastasis and circulating tumor cells. Circulating tumor cells (CTCs) were also found to be significantly reduced in DON-treated mice as compared to control, leading to reduced metastasis. Combination of low-dose DON (0.5mg/kg/3 days a week) with low-dose anti PD-1 (100 μgX3 times) caused an impressive reduction in tumor volume along with an increase in survival in KPC tumor-bearing mice. Flow-based analysis of tumor cells show a significant influx of CD8+ T cells along with an increase in M1 polarization of macrophages. Survival study of CD8 knockout mice (CD8KO), orthotopically implanted with KPC and fibroblast cells, show that at low doses DON loses its anticancer effect in absence of CD8 as demonstrated by decrease in survival in CD8KO mice as compared to tumor-bearing wild-type mice.
Conclusion: Novel therapeutic strategies that can make PDAC more amenable to immune therapy are urgently needed. In this context, our study is extremely timely as it targets specific metabolic pathways in cancer cells to overcome immune resistance and sensitize the tumor to anti-PD1 therapy.
Citation Format: Nikita S. Sharma, Vineet K. Gupta, Vanessa Garrido, Roey Hadad, Brittany Durden, Kousik Kesh, Anthony Ferrantella, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Targeting tumor-intrinsic metabolic node in pancreatic cancer causes tumor regression, remodels extracellular matrix, and sensitizes to anti-PD1 therapy abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B03.
Abstract
Pancreatic cancer remains a significant health burden associated with limited patient survival and poses a major therapeutic challenge. Among cancer cells, Cancer stem cells (CSCs) are ...instrumental in inducing chemoresistance and metastasis in pancreatic cancer. CD133 has been identified as a CSC surface marker in several malignancies including pancreatic cancer. However, the functional role of CD133 in CSCs still not clear. The present study investigates the role of caveolar lipid raft integrity in functional properties of CD133+ CSCs. Current study showed first time that CD133 localizes to caveolar lipid rafts in pancreatic cancer cells, and associates with Caveolin1 (Cav-1) and cholesterol to form an integral signaling complex which drives the downstream processes of chemoresistance and metastasis. Further analysis showed that the integrity of the lipid-raft is crucial for maintenance of the functional properties of pancreatic CSCs, and its disruption leads to increased chemo-sensitivity to chemotherapeutic compound paclitaxel both in vitro as well as in vivo. Additionally, disruption of lipid raft results in decreased invasiveness and metastatic ability of the cells by deregulation of FAK signaling. The study also reveals that pancreatic cancer cells that lack the CSC population marker, CD133, did not respond to lipid raft disruption. Our results indicated that targeting the lipid-raft integrity in pancreatic cancer can specifically eradicate the CD133+ CSCs in pancreatic tumors, leading to a better prognosis for the disease.
Citation Format: Vineet K. Gupta, Nikita S. Sharma, Kousik Kesh, Patrcia Dauer, Alice Nomura, Bhuwan Giri, Vikas Dudeja, Ashok Saluja, Sulagna Banerjee. Lipid raft integrity is essential for metastasis and chemoresistance properties of CD133+ cancer stem cells in pancreatic cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3551.
Background and Aims: We describe the epidemiological and clinical characteristics, and 28 day outcome of critically ill COVID-19 patients admitted to a tertiary care centre in India.
Material and ...Methods: We included 60 adult critically ill COVID-19 patients in this prospective observational study, admitted to the intensive care unit (ICU) after obtaining ethics committee approval and informed consent. Demographics, clinical data, and treatment outcome at 28 days were assessed.
Results: Demographic characteristics of the COVID-19 patients reveal that compared to the survivors, the non-survivors were significantly older 57.5 vs. 47.5 years, had more comorbid disease Charlson's comorbidity index 4 vs. 2, higher Apache II scores 19 vs. 8.5, and had significantly higher percentage of smokers. Diabetes mellitus and hypertension were the most common comorbidities. Dyspnea, fever, and cough were the most common presenting symptoms. Total leucocyte count as well as blood lactate level were significantly higher in non-survivors. Around 47% patients had severe ARDS, and 60% patients required invasive mechanical ventilation. 28 day ICU mortality was 50%, with a mortality of 75% in patients receiving invasive mechanical ventilation. Mortality was higher in males than females (57% vs. 33%). Acute kidney injury and septic shock were the most common non-pulmonary complications during ICU stay. Incidence of liver dysfunction, septic shock, and vasopressor use was significantly higher in the non-survivors.
Conclusion: This study demonstrates a high 28 day mortality in severe COVID-19 patients. Further well designed prospective studies with larger sample size are needed to identify the risk factors associated with poor outcome in such patients.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Adolescent mental health is a concern. A high magnitude of deranged mental health conditions among adolescents is prevailing, which often gets extended into adulthood too. Hence, assessment of mental ...health morbidities like depression, anxiety, and stress is crucial to address them. This study aimed to estimate the prevalence of depression, anxiety, and stress among school-going adolescents studying in classes IX-XII and find out its correlates among the subjects.
This cross-sectional study was conducted among 812 adolescents studying in classes IX-XII in four randomly selected schools of Haripal block of Hooghly district, West Bengal, in 2022. A complete enumeration of students in each class of the selected schools was done. Data were collected by using a pretested schedule that included standardized depression, anxiety, and stress scale (DASS-21). Descriptive statistics and a Chi-square test were applied. Regression analysis was doneto determine associations and compute the adjusted odds ratio. Data were analyzed using SPSS version 23.
Overall prevalence of depression, anxiety, and stress were found to be 52.3%, 47.4%, and 33.7% respectively. Subscale scores shows 16.1% and 10.8% suffered from extremely severe depression and anxiety, respectively. It was noted that these were more common among female students, the late adolescent age group (16-19 years), students having social problems in the family, and those reporting using substances.
The prevalence of depression, anxiety, and stress being high among school students indicates a significant burden of mental health disease. Effective strategies to alleviate the adverse mental health, along with early identification of disease, can help in along way.
Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This ...population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a "fail-safe" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the "stress" conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-non-coding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.
A videolaryngoscope has been recommended for intubation in the COVID-19 scenario but the videolaryngoscope providing optimal intubation conditions is not ascertained. We compared KingVision ...channelled blade with a non-Channelled videolaryngoscope for intubation times in a simulated COVID-19 intubation scenario by both anaesthesiologists and non-anaesthesiologists.
This prospective randomised cross over mannequin study was conducted in a skill training lab. 25 anaesthesiologists and 25 non-anaesthesiologists donned in standard personal protective equipment performed 100 intubations with KingVision and Tuoren videolaryngoscopes in a mannequin covered with a transparent plastic sheet. The total intubation time, percentage of glottic opening scores, first attempt success rates were assessed.
The mean difference in intubation times in anaesthesiologists and non-anaesthesiologist less with KingVision videolaryngoscope (21.1s; 95% CI 9.6–32.6s vs. 35.9s; 95% CI 24.4–47.4 s; P = 0.001). Percentage of glottic opening score was significantly better with KingVision by non-anaesthesiologists (60; IQR 42.5 to 75 vs. 70; IQR 50 to 100; P = 0.019). KingVision provided superior first attempt success rate in non-anaesthesiologists (84% vs. 61.9%; P = 0.02) and anaesthesiologists (96% vs. 76%; P = 0.12).
KingVision channelled videolaryngoscope provided faster intubation times, glottic views and first attempt success rates in a simulated COVID-19 scenario in manikins and might be preferred over videolaryngoscopes with non-channelled blade. The findings need to be further verified in humans.
ctri.nic.in identifier: REF/2020/05/033338.
•Intubation in COVID-19 scenario is challenging especially in hands of non-anaesthesiologists.•VL have been advocated for intubation in COVID-19 setting but specific type of VL to be used has not been ascertained yet.•We compared a channelled to a non-channelled blade VL in COVID scenario with respect to improvement in intubation conditions.•KingVision channelled VL reduced the intubation time and difficulty for both anaesthesiologists and non-anaesthesiologists.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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