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(1 - 5 of 5)
- Title
- A novel link between Akt1 and Twist1 in ovarian tumor cell motility and invasiveness.
- Creator
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Shah, Nirav, Altomare, Deborah, Zhao, Jihe, Khaled, Annette, University of Central Florida
- Abstract / Description
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Ovarian cancer results in more deaths per year than any other cancer of the female reproductive system. The low survival rate is partly due to the lack of early detection and the susceptibility to relapse. The AKT serine threonine kinase plays a pivotal role in hallmark cellular processes for the progression of ovarian cancer, including tumor cell growth and migration. Therapeutic targeting of pan-AKT has been problematic, in part due to feedback mechanisms and crosstalk with other pathways....
Show moreOvarian cancer results in more deaths per year than any other cancer of the female reproductive system. The low survival rate is partly due to the lack of early detection and the susceptibility to relapse. The AKT serine threonine kinase plays a pivotal role in hallmark cellular processes for the progression of ovarian cancer, including tumor cell growth and migration. Therapeutic targeting of pan-AKT has been problematic, in part due to feedback mechanisms and crosstalk with other pathways. The hypothesis for this study is that AKT 1, -2 and -3 isoforms may have different roles and regulate cell processes in uniquely varied ways. A transgenic mouse model that expresses the SV40 T-antigen viral oncogene and is known to have dramatically increased susceptibility to ovarian cancer was utilized, and it had genetic inactivation of either AKT1 or AKT2 through targeted deletion of the individual genes because these isoforms have been implicated in this cancer. Primary ovarian tumor cell cultures were established and found to exhibit different morphology, proliferation and migration that may indicate a different role for the AKT1 and AKT2 isoforms in these contexts. Ovarian tumor cells with absence of AKT1 predominantly exhibited reduced cell migration when compared to cells with retention of AKT1 and absence of AKT2. Since AKT is known to be important for epithelial-mesenchymal transition (EMT), a process potentially associated with tumor cell metastasis, the expression of transcription factors implicated in EMT was assessed by real-time array analysis in ovarian tumor cells knocked-out for either AKT1 or AKT2. Twist1, one of the major players in EMT, was not detectable in the cells missing the AKT1 isoform. Results indicate an association of Twist1 with AKT1 in EMT and migration of ovarian tumors cells. This finding is significant because AKT2 has been implicated as the major player of cell migration in human breast cancer cells. Collectively, these findings support a tissue specific role of the AKT isoforms, and may provide insights regarding the most useful cell context in order to target components of the AKT signaling pathway indirectly affecting EMT in order to prevent tumor progression in patients with ovarian and perhaps other types of cancers.
Show less - Date Issued
- 2012
- Identifier
- CFE0004630, ucf:49916
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004630
- Title
- TESTING MICE AT RISK OF PANCREATIC CANCER FOR ALTERED PROTEIN PATHWAYS FOUND IN DIABETES.
- Creator
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Cheung, Henley, Altomare, Deborah A., University of Central Florida
- Abstract / Description
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Pancreatic cancer is nearly asymptomatic, which can result in extensive grow and even metastasis to other organs before detection. When diagnosed at a late stage, the survival rate is 3%. Early detection is therefore the key to treating pancreatic cancer. Diabetes was identified as a risk factor for the development of pancreatic cancer, but the mechanism remains unknown. In this project, the objective was to delineate a link between diabetes and pancreatic cancer by examining their shared...
Show morePancreatic cancer is nearly asymptomatic, which can result in extensive grow and even metastasis to other organs before detection. When diagnosed at a late stage, the survival rate is 3%. Early detection is therefore the key to treating pancreatic cancer. Diabetes was identified as a risk factor for the development of pancreatic cancer, but the mechanism remains unknown. In this project, the objective was to delineate a link between diabetes and pancreatic cancer by examining their shared protein signaling pathways. In a previous study, hyper-activation of AKT1 resulted in a pre-diabetic phenotype and also increased upregulation of downstream phosphorylated mTOR and phosphorylated p70S6 kinase. More recently, mice with mutations that hyper-activated AKT1 and KRAS showed a significantly higher blood glucose level compared to littermate matched wild-type, mutant AKT1, or mutant KRAS mice. Interestingly, mice with a combination of mutations that hyper-activated AKT1 and KRAS also showed faster development of pancreatic cancer compared to these other groups of littermate mice. Toward determining a molecular basis for the crosstalk between AKT1 and KRAS, pancreas and liver tissues were collected from all four groups of mice including wild-type, mutant AKT1, mutant KRAS, and mice with dual AKT1/KRAS hyper-activation. One strategy was to examine expression and/or phosphorylation of downstream protein signaling crosstalk by analysis of p70S6K using Western Blots. Erk 1/2 proteins were also tested as downstream proteins of KRAS to provide a molecular view of the individual and cooperative roles of AKT1 and KRAS in the mouse models. A potential feedback mechanism to affect insulin receptor signaling in the pancreas was examined using enzyme-linked immunosorbent assays (ELISA). A significant decrease in insulin receptor phosphorylation, possibly contributing to insulin resistance, was found when mice had mutant hyper-activated KRAS. Contrary to the original expectations, mice with combined mutations of AKT1 and KRAS may contribute to the accentuated diabetic phenotype by targeting two different points in the AKT and KRAS protein signaling pathways. The information can help understand the relationship between glucose metabolism, diabetes, and pancreatic cancer development. By thoroughly studying the interactions between targets in the AKT1/KRAS signaling pathways, key molecular events that induce metabolic changes and potentially early biomarkers may lead to an improved understanding of risk and/or detection of pancreatic cancer.
Show less - Date Issued
- 2017
- Identifier
- CFH2000273, ucf:45895
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH2000273
- Title
- MANIPULATING AKTIVATED METABOLISM VIA MTORC1.
- Creator
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von Hack-Prestinary, Ivan, Altomare, Deborah, University of Central Florida
- Abstract / Description
-
Although poorly understood, normal cells and cancerous cells of the same type exhibit different patterns of nutrient consumption, processing and utility of metabolic substrates. Differences in substrate uptake, preference, and alternately emphasized metabolic pathways offer opportunities for selective targeting of cancer versus stroma. This may be accomplished by using a sequential approach of nutrient deprivation and pharmaceutical perturbation of metabolic pathways to inhibit cellular...
Show moreAlthough poorly understood, normal cells and cancerous cells of the same type exhibit different patterns of nutrient consumption, processing and utility of metabolic substrates. Differences in substrate uptake, preference, and alternately emphasized metabolic pathways offer opportunities for selective targeting of cancer versus stroma. This may be accomplished by using a sequential approach of nutrient deprivation and pharmaceutical perturbation of metabolic pathways to inhibit cellular proliferation. The purpose of this study was to investigate the effects of restricting glucose and glutamine concentrations, in vitro, to levels that resemble a potential human fasting state. The mammalian target of rapamycin (mTOR), a mediator of nutrient sensation, was then inhibited with rapamycin in the nutrient-restricted conditions. Because active Akt/mTOR is implicated in cancer cell pro-survival, the hypothesis is that pharmaceutical inhibition of active Akt/mTOR signaling in combination with the stress of restricted nutrient supply will be more effective than nutrient deprivation alone at disrupting metabolic processes to impair cancer cell proliferation and/or pro-survival mechanisms. Untreated and treated conditions were tested to determine if an additive or synergistic effect would result from a sequential insult of nutrient deprivation followed by inhibited mTORC1 signaling. The cell line used for this study was cultivated from a murine pancreatic intraepithelial neoplasia (PANIN) derived from a transgenic mouse with pancreatic tissue-specific expression of constitutively active Akt. The transgene of Akt, isoform 1, contains a myristoyl tag that facilitates co-localization of Akt to the plasma membrane, thereby promoting the activation of this signaling protein. This aberrantly activated Akt represents a prosurvival condition observed in most cancers, and impacts metabolic balance with increased downstream signaling to metabolic sensors and regulators, including mTORC1. Several methods were used to evaluate changes in metabolic and physiological response to nutrient deprivation and mTORC1 inhibition. These included tetrazolium reduction/absorbance readings to qualitatively evaluate differences in cell proliferation, and Western immunoblots for observing changes in protein expression and phosphorylation. ATP luminescence assays were applied to quantify intracellular ATP content, and citrate synthase spectrophotometry used to quantify specific activity/indicate changes in the TCA/OXPHOS production of ATP. Results from the above methods suggest that, individually, nutrient deprivation and rapamycin treatment share some similar effects on metabolically-related protein phosphorylation and in reducing cellular proliferation. Collectively, nutrient deprivation plus rapamycin treatment, however, resulted in unanticipated metabolic alterations under conditions used for this study, the complexities of which would need to be delineated in future studies.
Show less - Date Issued
- 2013
- Identifier
- CFH0004373, ucf:45006
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFH0004373
- Title
- Bone Morphogenetic Protein-7 (BMP-7) Polarizes Monocytes into M2 Macrophages.
- Creator
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Rocher, Crystal, Singla, Dinender, Siddiqi, Shadab, Sugaya, Kiminobu, University of Central Florida
- Abstract / Description
-
Atherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however,...
Show moreAtherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however, its role in monocyte polarization is yet to be revealed. In the current study, we hypothesize that monocyte treatment with BMP-7 will significantly result in increased polarization of monocytes into M2 macrophages and increased expression of anti-inflammatory cytokines. To that effect, we have established a stress induced cell culture system with monocytes (THP-1 cells) and apoptotic conditioned medium (ACM), simulating injury, to understand the effects of BMP-7 on M2 macrophage polarization from monocytes. Our data demonstrates that the BMP type 2 receptor (BMPR2) is found on monocytes and its activation is significantly (p(<)0.05) increased in both monocytes and M2 macrophages following treatment with BMP-7. Furthermore, a significant (p(<)0.05) increase of M2 macrophages in the BMP-7 treated group was shown following immunostaining with CD206 and arginase-1, two M2 macrophage markers, whereas a significant (p(<)0.05) decrease of iNOS expression, an M1 macrophage marker, was shown. Moreover, treatment with BMP-7 resulted in significantly (p(<)0.05) increased expression of IL-10 and IL-1ra, two anti-inflammatory cytokines, but significantly (p(<)0.05) decreased levels of the pro-inflammatory cytokines, MCP-1, IL-6 and TNF-?. We also hypothesize that polarization of monocytes to M2 macrophages occurs through activation of SMAD1/5/8 and PI3K-Akt-mTOR pathways. Upon BMP-7 binding to its receptor, BMPR2, activation of SMAD1/5/8 occurs which then activates the p85 subunit of PI3K resulting in downstream activation of Akt and mTOR. Our data shows that following treatment with BMP-7, expression of p-SMAD1/5/8, p-PI3K, p-Akt and p-mTOR is significantly (p(<)0.05) increased compared to controls whereas p-PTEN, an inhibitor of the PI3K pathway, is significantly (p(<)0.05) decreased in the BMP-7 treated group compared to controls. In conclusion, our data reveals that BMP-7 polarizes monocytes into M2 macrophages and it achieves this through activation of the PI3K-Akt-mTOR pathway, which will have significant applications for atherosclerosis treatment.
Show less - Date Issued
- 2013
- Identifier
- CFE0004922, ucf:49617
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004922
- Title
- Diabetes Phenotypes in Transgenic Pancreatic Cancer Mouse Models.
- Creator
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Albury, Toya, Altomare, Deborah, Zhao, Jihe, Masternak, Michal, Khaled, Annette, University of Central Florida
- Abstract / Description
-
Protein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in...
Show moreProtein Kinase B/AKT, a serine/threonine kinase with three isoforms (AKT1-3), is downstream of phosphatidylinositol 3-kinase (PI3K), and signals through the phosphorylation and subsequent activation or inhibition of downstream substrates, such as mammalian target of rapamycin complex 1 (mTORC1) or glycogen synthase kinase 3 beta (GSK-3?), respectively. The AKT1 isoform is predominantly recognized for regulation of cell survival, growth, and proliferation, due to its constitutive activation in pancreatic cancers (e.g., islet cell carcinoma and pancreatic adenocarcinoma). The progression of pancreatic ductal adenocarcinoma (PDAC), the most lethal common cancer, is initiated by activation mutations of the KRas oncogene. This leads to additional molecular changes, such as activation of the AKT1 oncogene, which drives PDAC progression and tumor formation. By mating transgenic mice with activation of KRas (Pdx- Cre;LSL-KRasG12D) and mice with activation of AKT1 (Pdx- Tta;TetO-MyrAKT1) we were able to produce mice with two activated oncogenes (AKT1Myr/KRasG12D) for comparative studies. Kaplan-Meier survival curves, histology, and genomic/proteomic analysis were used to characterize the incidence and frequency of histological (e.g. presence of mucin-4 in pancreatic intraepithelial neoplasms) and genetic (e.g. loss of tumor suppressors p16Ink4a and p19Arf) alterations known to commonly occur in human pancreatic cancer, as well as delineate the role of AKT1 in accelerating pancreatic tumor progression and metastasis. We determined that AKT1Myr/KRasG12D mice, unlike other PDAC mouse models, accurately mimic the human PDAC progression molecularly, structurally, and temporally. Interestingly, the AKT1Myr and AKT1Myr/KRasG12D models both exhibit a pre-tumor, diabetic phenotype. While, AKT1 hyperactivation in various cancers has been thoroughly studied, its role in glucose metabolism has been noted, but comparatively overlooked. As early as the 1900s a relationship between diabetes and pancreatic cancer has been proposed. With 80% of PDAC patients suffering from hyperglycemia or diabetes prior to diagnosis, one prevailing theory is that new onset diabetes is an early marker for pancreatic cancer. This is also supported by experimental and clinical studies, such as the resolution of diabetes with tumor removal and the induction of hyperglycemia with the implantation of cancer cell lines. To better understand the role of AKT1 and its hyperactivation in glucose metabolism, AKT1Myr mice were characterized via metabolic (e.g. glucose/insulin tolerance test) and histological (e.g. immunohistochemistry) studies. Beginning at weaning, 3 weeks of age, the glucose intolerant AKT1Myr mice exhibited non-fasted hyperglycemia, which progressed to fasted hyperglycemia by 5 months of age. The glucose intolerance was attributed to a fasted hyperglucagonemia, and hepatic insulin resistance detectable by reduced phosphorylation of the insulin receptor following insulin injection into the inferior vena cava. Additionally, AKT1Myr/KRasG12D mice currently being studied, appear to display a more severe diabetic phenotype, with fasted hyperglycemia noticeable at an earlier age, fasted hyperglucagonemia, polyuria, muscle wasting, and bloating. Treatment of both models with doxycycline diet, to turn-off the transgene, caused attenuation of the non-fasted and fasted hyperglycemia, thus affirming AKT1 hyperactivation as the trigger. These newly revealed roles of AKT1, along with future studies of these mouse models, will better delineate the molecular mechanisms responsible for the individual and joint roles of AKT1 and KRas in pancreatic cancer oncogenesis, the initiation of cancer associated diabetes, and the association of these two diseases.
Show less - Date Issued
- 2015
- Identifier
- CFE0006245, ucf:51081
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006245
