2003 |
Fatatis, Alessandro |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Intracellular Sphingolipids in Calcium Signaling
[unreadable] DESCRIPTION (provided by applicant): [unreadable] AIterations in calcium (Ca) signaling are implicated in human pathologies ranging from neoplastic to neurodegenerative diseases. The spatio-temporal characteristics of Ca signals can regulate the activity of transcription factors and directly affect gene expression. Ca is generally mobilized from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which activates specific receptors containing an intrinsic Ca channel (InsP3Rs) and localized to the endoplasmic reticulum. Notably, many cell-surface receptors that increase InsP3 concurrently increase the intracellular production of sphingosine and sphingosine-1 phosphate (sphingosine-lP) as well. A Ca-mobilizing role for these two sphingolipids has been proposed, but their mechanism of action is unclear and the intracellular Ca channel(s) they activate yet to be identified. Furthermore, their functional interactions with InsP3 have not been characterized. The current proposal aims to test the hypothesis that both intracellular sphingosine and sphingosine-lP mobilize Ca through InsP3Rs. The specific aims of this proposal are: 1) To characterize the Ca-mobilizing action of intracellular sphingosine and sphingosine-lP - acting separately or in combination with InsP3 - using caged-precursors that will be photo-activated inside living cells and their effects analyzed by single-cell microfluorimetry and digital imaging. The reciprocal interactions between InsP3 and the two sphingolipids during the Ca signaling following the stimulation of several plasma membrane receptors will also be investigated; 2) To establish whether the expression of different InsP3R-subtypes can determine the type of Ca signals evoked by intracellular sphingosine and sphingosine-lP. Experiments using cells genetically engineered to express specific InsP3R-subtypes will be combined with studies performed with mammalian cell lines transfected with distinct InsP3R-subtypes; 3) To ascertain whether sphingosine and sphingosine-lP directly open the InsP3R-channel and/or functionally modulate the action of InsP3. To this end, single-channel recording from native and recombinant InsP3R subtypes reconstituted in planar lipid bilayers will be employed. The long-term objective of this proposal is to identify the mechanisms and intracellular channels through which sphingosine and sphingosine-lP mobilize intracellular Ca - acting alone or in combination with InsP3 - and characterize their role in cellular signaling following the stimulation of plasma membrane receptors. [unreadable] [unreadable]
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1 |
2004 — 2007 |
Fatatis, Alessandro |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Intracellular Sphingolipids in Calcium Signaling
[unreadable] DESCRIPTION (provided by applicant): [unreadable] AIterations in calcium (Ca) signaling are implicated in human pathologies ranging from neoplastic to neurodegenerative diseases. The spatio-temporal characteristics of Ca signals can regulate the activity of transcription factors and directly affect gene expression. Ca is generally mobilized from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which activates specific receptors containing an intrinsic Ca channel (InsP3Rs) and localized to the endoplasmic reticulum. Notably, many cell-surface receptors that increase InsP3 concurrently increase the intracellular production of sphingosine and sphingosine-1 phosphate (sphingosine-lP) as well. A Ca-mobilizing role for these two sphingolipids has been proposed, but their mechanism of action is unclear and the intracellular Ca channel(s) they activate yet to be identified. Furthermore, their functional interactions with InsP3 have not been characterized. The current proposal aims to test the hypothesis that both intracellular sphingosine and sphingosine-lP mobilize Ca through InsP3Rs. The specific aims of this proposal are: 1) To characterize the Ca-mobilizing action of intracellular sphingosine and sphingosine-lP - acting separately or in combination with InsP3 - using caged-precursors that will be photo-activated inside living cells and their effects analyzed by single-cell microfluorimetry and digital imaging. The reciprocal interactions between InsP3 and the two sphingolipids during the Ca signaling following the stimulation of several plasma membrane receptors will also be investigated; 2) To establish whether the expression of different InsP3R-subtypes can determine the type of Ca signals evoked by intracellular sphingosine and sphingosine-lP. Experiments using cells genetically engineered to express specific InsP3R-subtypes will be combined with studies performed with mammalian cell lines transfected with distinct InsP3R-subtypes; 3) To ascertain whether sphingosine and sphingosine-lP directly open the InsP3R-channel and/or functionally modulate the action of InsP3. To this end, single-channel recording from native and recombinant InsP3R subtypes reconstituted in planar lipid bilayers will be employed. The long-term objective of this proposal is to identify the mechanisms and intracellular channels through which sphingosine and sphingosine-lP mobilize intracellular Ca - acting alone or in combination with InsP3 - and characterize their role in cellular signaling following the stimulation of plasma membrane receptors. [unreadable] [unreadable]
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1 |
2016 — 2020 |
Fatatis, Alessandro Meucci, Olimpia (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Cx3cr1 in Breast Cancer Metastasis
Breast cancer patients die from metastatic disease. Primary breast adenocarcinoma spreads to distant organs by shedding circulating tumor cells (CTCs) in the blood. When these cells leave the systemic circulation, they convert into Disseminated Tumor Cells (DTCs), which are the seeds of secondary tumors. Mounting evidence indicates that existing metastases can mobilize cancer cells back into the blood, thus leading to further spreading that precipitates clinical progression. Thus, strategies aimed to impair tumor seeding have the potential to arrest or significantly decelerating the extension of the disease. Chemokine receptors have been implicated in dissemination, proliferation and survival of cancer cells. Our recent work indicates that the chemokine receptor CX3CR1 is over-expressed in both primary breast tumors and metastatic lesions and plays a role in the lodging of breast cancer cells to the skeleton of animal models. Furthermore, studies form others have shown that this receptor is expressed by cells with tumor-forming abilities and can transactivate growth factor receptors regulating proliferation and survival. Thus, based on the existing literature and our preliminary data, we hypothesize that CX3CR1 is implicated in both early and late stages of metastasis and that its blockade can both deter breast cancer cells from seeding multiple organs and counter their tumor- initiating properties. To test this hypothesis, we will use a combination of CRISPR interference (CRISPRi) and a novel small-molecule antagonist to delineate the time-frame for CX3CR1 involvement in metastatic progression and the effects of pharmacologic targeting of CX3CR1 in tertiary spreading from existing metastases. Finally, we intend to gain a mechanistic understanding of CX3CR1 signaling and determine the molecular pathways associated with tumor-initiation properties activated by this receptor in breast cancer cells. This proposal is structured into three specific aims: AIM 1. To delineate the temporal involvement of CX3CR1 in metastatic progression; AIM 2. To define the effects of CX3CR1 blockade on tumor re- seeding from existing metastases; AIM 3. To elucidate the mechanistic details of CX3CR1 involvement in the metastatic behavior of breast cancer cells. This proposal will define how CX3CR1 influences breast cancer metastatic behavior, reveal important mechanistic details of its activity in cancer cells and provide pre-clinical support for the pharmacologic targeting of this receptor, which presents a high therapeutic potential from several standpoints. From a drug-safety perspective, transgenic mice knockout for CX3CR1 are viable and exhibit no impairment of their immune response under unchallenged conditions, overt behavioral abnormalities or macroscopic anatomical alterations. Thus, if successful our work should pave the way to a novel series of therapeutics and promote multifaceted strategies to counteract the metastatic progression of breast cancer.
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1 |