The Roberts lab is defining functions of the modulatory extracellular matrix protein thrombospondin-1, its receptors, and downstream signal transduction pathways in cancer. We have identified functions for thrombospondin-1 and its cell surface signaling receptor CD47 in regulating tumor angiogenesis, perfusion, and antitumor immunity. CD47 signaling also limits recovery of animals from stress induced by ischemia, ionizing radiation, and cytotoxic chemotherapy. CD47 expressed by cancer cells and by cells in the tumor microenvironment can limit patient responses to therapy and directly promote tumor growth. Based on these insights, we have developed novel therapeutics approaches to target CD47 that enhance the efficacy of conventional chemotherapy, radiation therapy, and immunotherapy in murine cancer models. Address: Building 10 Room 2S235 10 Center Drive Bethesda, MD 20892-1500
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expr... more Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc, Induction of committed erythroid precursors is consistent with the known function of CD47 to l...
Journal of Cell Communication and Signaling, Jan 7, 2021
Targeted gene disruption in mice has provided valuable insights into the functions of matricellul... more Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.
Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhanc... more Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.Electronic supplementary materialThe online version of this article (10.1007/s00262-019-02397-7) contains supplementary material, which is available to authorized users.
The regulation of mucin secretion by SW1116 human colon carcinoma cells has been studied using mo... more The regulation of mucin secretion by SW1116 human colon carcinoma cells has been studied using monoclonal antibody 19-9, which has previously been used to detect mucin in the serum of cancer and cystic fibrosis patients. We found that SW1116 cells constitutively secrete considerable amounts of mucin as the predominant glycoprotein. The secretion of mucin by these cells is independent of cyclic AMP levels, but can be further stimulated by the Ca2+ ionophore A23187. However, arachidonic acid and its metabolites inhibit mucin secretion. Electron microscope studies reveal that the mucin is located near the plasma membrane as well as in vesicular and lysosome-like structures. However, the secretion pathway of mucin is different than that of the lysosomal contents, since arachidonic acid, while inhibiting mucin secretion, actually activates the secretion of the lysosomal enzyme beta-glucuronidase. We suggest that the mechanism of mucin secretion by SW1116 cells occurs by a pathway differe...
Components of the extracellular matrix have been shown to modulate the interaction of endothelial... more Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin re...
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expr... more Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc, Induction of committed erythroid precursors is consistent with the known function of CD47 to l...
Journal of Cell Communication and Signaling, Jan 7, 2021
Targeted gene disruption in mice has provided valuable insights into the functions of matricellul... more Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.
Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhanc... more Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47 m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47 m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47 m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.Electronic supplementary materialThe online version of this article (10.1007/s00262-019-02397-7) contains supplementary material, which is available to authorized users.
The regulation of mucin secretion by SW1116 human colon carcinoma cells has been studied using mo... more The regulation of mucin secretion by SW1116 human colon carcinoma cells has been studied using monoclonal antibody 19-9, which has previously been used to detect mucin in the serum of cancer and cystic fibrosis patients. We found that SW1116 cells constitutively secrete considerable amounts of mucin as the predominant glycoprotein. The secretion of mucin by these cells is independent of cyclic AMP levels, but can be further stimulated by the Ca2+ ionophore A23187. However, arachidonic acid and its metabolites inhibit mucin secretion. Electron microscope studies reveal that the mucin is located near the plasma membrane as well as in vesicular and lysosome-like structures. However, the secretion pathway of mucin is different than that of the lysosomal contents, since arachidonic acid, while inhibiting mucin secretion, actually activates the secretion of the lysosomal enzyme beta-glucuronidase. We suggest that the mechanism of mucin secretion by SW1116 cells occurs by a pathway differe...
Components of the extracellular matrix have been shown to modulate the interaction of endothelial... more Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin re...
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