Periodic Reporting for period 4 - Secret Surface (The cell surface tetraspanin web drives tumour development and alters metabolic signalling)
Okres sprawozdawczy: 2022-04-01 do 2023-03-31
Cancer development is characterized by uncontrolled proliferation, cell survival and metabolic reprogramming. Tumour cells are surrounded by a fluid-mosaic membrane that contains tetraspanins (Tspans) which are evolutionary conserved proteins important in the organisation of the cell surface. Increasing evidence indicates that Tspans are involved in cancer, still the mechanisms in native tumour membranes and its (patho)physiological functions have not been resolved. In this ERC project, we hypothesized that tumour cells contain a disrupted cell surface organisation leading to aberrant metabolic signalling and tumour development. The overall objective of Secret Surface is to unravel the composition, physiological functions and molecular mechanisms of the Tspan web on tumour development and clinical outcome. We focused on studying lymphomas using 3 subaims: I. Detailed analyses of Tspan web composition in lymphoma to select clinically relevant Tspans. II. Resolve the endogenous Tspan web on lymphoma cells and generation and analysis of lymphoma cells that have a complete deficiency of multiple Tspans. III. Decipher molecular mechanisms underlying Tspan web function in lymphoma cells. This project resulted in new insight into Tspan function in cancer that affects signalling pathways of lymphomas, which has implications for development of new cancer therapies.
The overall aim of Secret Surface is to unravel the composition, physiological functions and underlying molecular mechanisms of the cell surface tetraspanin web on tumour behaviour and clinical outcome in cancer patients.
Aim I. Tetraspanin web on lymphoma. We investigated tetraspanin expression at the protein level in a discovery cohort of human diffuse large B cell lymphoma (DLBCL) tissues using tissue microarray technology.
We identified a novel Tspan to be differentially expressed in lymphoma tissues, in contrast to other multiple other Tspan tested. Protein expression of this Tspan was directly related to subtype of lymphoma (ABC-DLBCL) in a patient cohort. We identified a new interaction of this Tspan with the adhesion receptor CD18 in the membrane of lymphoma cells. These data identify a novel biomarker in human DLBCL and we are currently investigating the underlying molecular mechanisms (unpublished data).
Aim II. Resolving the endogenous Tspan web on healthy and lymphoma B cells.
We successfully set-up dSTORM super-resolution microscopy of tetraspanin CD9 in B cell lines enabling the accurate analysis of the large data sets in terms of cluster size, diameter and number molecules/cluster. Using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation. Our results have just been published in FEBS Journal (DOI: 10.1111/febs.15295).
Recent studies show that Tspans can exist in an open and closed conformation. Thus, we generated mutants of Tspans (CD81 and CD53) and analysed organization in lymphoma B cells using super-resolution imaging. Our preliminary data show that the mutants differ in their capacity to interact with partner proteins in the cell surface of membrane proteins. In addition, we observed that glycosylation of the Tspans affects interaction with other membrane proteins. These studies provide more insight into the molecular basis of the Tspan web at the cell surface of healthy and malignant B cells (unpublished).
Aim III. Deciphering molecular mechanisms underlying Tspan web function in lymphoma cells
Using an unbiased metabolomics approach, we discovered that lymphoma B-cells deficient in tetraspanin CD37 have altered metabolic pathways resulting in increased fatty acid oxidation and higher ATP production. We confirmed that tetraspanin-deficient lymphoma cells responded higher to fatty acid stimulation by generating significantly more ATP and consuming more oxygen than tetraspanin-positive lymphoma cell. Strikingly, inhibition of the mitochondrial fatty acid transporter resulted in significantly more cell death in tetraspanin-negative lymphomas. These results demonstrate a functional metabolic switch DLBCL that may be exploited for future clinical intervention. These studies have been published in Nat Commun. (doi: 10.1038/s41467-022-33138-7).
Aim I. Tetraspanin web on lymphoma. We investigated tetraspanin expression at the protein level in a discovery cohort of human diffuse large B cell lymphoma (DLBCL) tissues using tissue microarray technology.
We identified a novel Tspan to be differentially expressed in lymphoma tissues, in contrast to other multiple other Tspan tested. Protein expression of this Tspan was directly related to subtype of lymphoma (ABC-DLBCL) in a patient cohort. We identified a new interaction of this Tspan with the adhesion receptor CD18 in the membrane of lymphoma cells. These data identify a novel biomarker in human DLBCL and we are currently investigating the underlying molecular mechanisms (unpublished data).
Aim II. Resolving the endogenous Tspan web on healthy and lymphoma B cells.
We successfully set-up dSTORM super-resolution microscopy of tetraspanin CD9 in B cell lines enabling the accurate analysis of the large data sets in terms of cluster size, diameter and number molecules/cluster. Using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation. Our results have just been published in FEBS Journal (DOI: 10.1111/febs.15295).
Recent studies show that Tspans can exist in an open and closed conformation. Thus, we generated mutants of Tspans (CD81 and CD53) and analysed organization in lymphoma B cells using super-resolution imaging. Our preliminary data show that the mutants differ in their capacity to interact with partner proteins in the cell surface of membrane proteins. In addition, we observed that glycosylation of the Tspans affects interaction with other membrane proteins. These studies provide more insight into the molecular basis of the Tspan web at the cell surface of healthy and malignant B cells (unpublished).
Aim III. Deciphering molecular mechanisms underlying Tspan web function in lymphoma cells
Using an unbiased metabolomics approach, we discovered that lymphoma B-cells deficient in tetraspanin CD37 have altered metabolic pathways resulting in increased fatty acid oxidation and higher ATP production. We confirmed that tetraspanin-deficient lymphoma cells responded higher to fatty acid stimulation by generating significantly more ATP and consuming more oxygen than tetraspanin-positive lymphoma cell. Strikingly, inhibition of the mitochondrial fatty acid transporter resulted in significantly more cell death in tetraspanin-negative lymphomas. These results demonstrate a functional metabolic switch DLBCL that may be exploited for future clinical intervention. These studies have been published in Nat Commun. (doi: 10.1038/s41467-022-33138-7).
WPI. Tspan web in lymphoma patients. Detailed analyses of Tspan web composition in lymphoma and clinical outcome in patients through high-throughput tissue microarray technology and multispectral imaging. We identified a novel tumour suppressor protein in diffuse large B cell lymphoma. We have tissue microarrays available of an independent cohort of DLBCL patients to confirm that tetraspanin expression is correlated to clinical outcome (overall and progression-free survival). Current research focusses on identifying the molecular mechanism underlying tetraspanin function in lymphoma cells. We developed CRISPR-Cas9 primers to knock-out tetraspanins in different B cell lines which will be used to analyse cell survival, adhesion migration and metastasis.
WPII. Modulation and characterisation of the Tspan web. Based on our recently published work in which we established super-resolution imaging of tetraspanin CD9 on lymphoma cell lines.
We will extend our advanced imaging studies to other tetraspanins and their partner proteins within the cell surface of lymphoma versus healthy B cells. In addition, we also initiated studies to decipher the function of the tetraspanin-like protein CD20. CD20 is the target of rituximab which is together with chemotherapy (CHOP) the standard of care treatment for patients with mature B cell tumours. Moreover, we have generated lymphoma cells that lack two tetraspanins that will be used for functional characterization (proliferation, cytokine production etc), and we aim to knock out more B cell tetraspanins within the same cells (CRISPR/Cas9 technology). I expect that these studies will provide valuable new insight into the function of tetraspanins B cells, including the primary target of rituximab CD20, which may facilitate future (immune-based) therapies for patients with aggressive B cell lymphoma.
WPIII. Tspan-mediated signalling mechanisms. Deciphering molecular mechanisms underlying Tspan web function in lymphoma cells, and validation in selected preclinical mouse models.
Using an unbiased metabolomics approach, we discovered that lymphoma B-cells deficient in tetraspanins have altered metabolic pathways resulting in increased fatty acid oxidation and higher ATP production. We are currently investigating the underlying molecular mechanisms in lymphoma cells. We expect these results to demonstrate a functional metabolic switch in DLBCL that may be exploited for future clinical intervention.
WPII. Modulation and characterisation of the Tspan web. Based on our recently published work in which we established super-resolution imaging of tetraspanin CD9 on lymphoma cell lines.
We will extend our advanced imaging studies to other tetraspanins and their partner proteins within the cell surface of lymphoma versus healthy B cells. In addition, we also initiated studies to decipher the function of the tetraspanin-like protein CD20. CD20 is the target of rituximab which is together with chemotherapy (CHOP) the standard of care treatment for patients with mature B cell tumours. Moreover, we have generated lymphoma cells that lack two tetraspanins that will be used for functional characterization (proliferation, cytokine production etc), and we aim to knock out more B cell tetraspanins within the same cells (CRISPR/Cas9 technology). I expect that these studies will provide valuable new insight into the function of tetraspanins B cells, including the primary target of rituximab CD20, which may facilitate future (immune-based) therapies for patients with aggressive B cell lymphoma.
WPIII. Tspan-mediated signalling mechanisms. Deciphering molecular mechanisms underlying Tspan web function in lymphoma cells, and validation in selected preclinical mouse models.
Using an unbiased metabolomics approach, we discovered that lymphoma B-cells deficient in tetraspanins have altered metabolic pathways resulting in increased fatty acid oxidation and higher ATP production. We are currently investigating the underlying molecular mechanisms in lymphoma cells. We expect these results to demonstrate a functional metabolic switch in DLBCL that may be exploited for future clinical intervention.