Publications
Selected Publications
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Breanna Caruso, Amy E. Moran, Thymic expression of immune checkpoint molecules and their implication for response to immunotherapies, Trends in Cancer, 2023, ISSN 2405-8033, https://doi.org/10.1016/j.trecan.2023.04.007.
Abstract
The thymus is responsible for generating a diverse T cell repertoire that is tolerant to self, but capable of responding to various immunologic insults, including cancer. Checkpoint blockade has changed the face of cancer treatment by targeting inhibitory molecules, which are known to regulate peripheral T cell responses. However, these inhibitory molecules and their ligands are expressed during T cell development in the thymus. In this review, we describe the underappreciated role of checkpoint molecule expression during the formation of the T cell repertoire and detail the importance of inhibitory molecules in regulating T cell lineage commitment. Understanding how these molecules function in the thymus may inform therapeutic strategies for better patient outcomes.
The thymus is responsible for generating a diverse T cell repertoire that is tolerant to self, but capable of responding to various immunologic insults, including cancer. Checkpoint blockade has changed the face of cancer treatment by targeting inhibitory molecules, which are known to regulate peripheral T cell responses. However, these inhibitory molecules and their ligands are expressed during T cell development in the thymus. In this review, we describe the underappreciated role of checkpoint molecule expression during the formation of the T cell repertoire and detail the importance of inhibitory molecules in regulating T cell lineage commitment. Understanding how these molecules function in the thymus may inform therapeutic strategies for better patient outcomes.
Polesso F, Caruso B, Hammond SA, Moran AE. Restored Thymic Output after Androgen Blockade Participates in Antitumor Immunity. J Immunol. 2023 Feb 15;210(4):496-503. doi: 10.4049/jimmunol.2200696. PMID: 36548468; PMCID: PMC9898132.
Abstract
The thymus is a hormone-sensitive organ, which involutes with age in response to production of sex steroids. Thymic involution leads to a decrease in the generation of recent thymic emigrants (RTEs), resulting in a reduced response to immune challenges such as cancer. Interestingly, the standard of care for prostate cancer patients is androgen deprivation therapy (ADT), which leads to thymic regeneration and an increase in thymic output. It remains unknown whether these newly produced T cells can contribute to the antitumor immune response. This study defines the kinetics of thymic regeneration in response to ADT in mice, determining that thymic epithelial cell proliferation is critical for the increase in RTE output. Using a mouse model to track RTE in vivo, we demonstrate that these newly generated RTEs can traffic to tumors, where they become activated and produce effector cytokines at levels similar to more mature T cells. Collectively, these data suggest that RTEs produced from ADT-induced thymic regeneration could be harnessed for the antitumor immune response.
The thymus is a hormone-sensitive organ, which involutes with age in response to production of sex steroids. Thymic involution leads to a decrease in the generation of recent thymic emigrants (RTEs), resulting in a reduced response to immune challenges such as cancer. Interestingly, the standard of care for prostate cancer patients is androgen deprivation therapy (ADT), which leads to thymic regeneration and an increase in thymic output. It remains unknown whether these newly produced T cells can contribute to the antitumor immune response. This study defines the kinetics of thymic regeneration in response to ADT in mice, determining that thymic epithelial cell proliferation is critical for the increase in RTE output. Using a mouse model to track RTE in vivo, we demonstrate that these newly generated RTEs can traffic to tumors, where they become activated and produce effector cytokines at levels similar to more mature T cells. Collectively, these data suggest that RTEs produced from ADT-induced thymic regeneration could be harnessed for the antitumor immune response.
Guan X, Polesso F, Wang C, Sehrawat A, Hawkins RM, Murray SE, Thomas GV, Caruso B, Thompson RF, Wood MA, Hipfinger C, Hammond SA, Graff JN, Xia Z, Moran AE. (2022). Androgen receptor activity in T cells limits checkpoint blockade efficacy. Nature. 23 March 2022. PMID: 35322234
Abstract
Immune checkpoint blockade has revolutionized the field of oncology, inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer, immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth, and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFNγ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together, our findings establish that T cell intrinsic AR activity represses IFNγ expression and represents a novel mechanism of immunotherapy resistance.
Immune checkpoint blockade has revolutionized the field of oncology, inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer, immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth, and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFNγ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together, our findings establish that T cell intrinsic AR activity represses IFNγ expression and represents a novel mechanism of immunotherapy resistance.
Vellano CP, White MG, Andrew MC, Chelvanambi M, Witt RG, Daniele JR, et al, Moran AE, Wang L, Heffernan T, Marszalek J, Wargo JA. (2022). Androgen receptor blocakde promotes response to BRAF/MEK-targeted therapy. Nature. In press, March 2022.
Abstract
Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.
Treatment with therapy targeting BRAF and MEK (BRAF/MEK) has revolutionized care in melanoma and other cancers; however, therapeutic resistance is common and innovative treatment strategies are needed. Here we studied a group of patients with melanoma who were treated with neoadjuvant BRAF/MEK-targeted therapy ( NCT02231775 , n = 51) and observed significantly higher rates of major pathological response (MPR; ≤10% viable tumour at resection) and improved recurrence-free survival (RFS) in female versus male patients (MPR, 66% versus 14%, P = 0.001; RFS, 64% versus 32% at 2 years, P = 0.021). The findings were validated in several additional cohorts2-4 of patients with unresectable metastatic melanoma who were treated with BRAF- and/or MEK-targeted therapy (n = 664 patients in total), demonstrating improved progression-free survival and overall survival in female versus male patients in several of these studies. Studies in preclinical models demonstrated significantly impaired anti-tumour activity in male versus female mice after BRAF/MEK-targeted therapy (P = 0.006), with significantly higher expression of the androgen receptor in tumours of male and female BRAF/MEK-treated mice versus the control (P = 0.0006 and P = 0.0025). Pharmacological inhibition of androgen receptor signalling improved responses to BRAF/MEK-targeted therapy in male and female mice (P = 0.018 and P = 0.003), whereas induction of androgen receptor signalling (through testosterone administration) was associated with a significantly impaired response to BRAF/MEK-targeted therapy in male and female patients (P = 0.021 and P < 0.0001). Together, these results have important implications for therapy.
Sun D, Guan X, Moran AE, Wu LY, Qian DZ, Schedin P, Dai MS, Danilov AV, Alumkal JJ, Adey AC, Spellman PT, Xia Z. Identifying phenotype-associated subpopulations by integrating bulk and single-cell sequencing data. Nat Biotechnol. 2022 Apr;40(4):527-538. doi: 10.1038/s41587-021-01091-3. Epub 2021 Nov 11. PMID: 34764492; PMCID: PMC9010342.
Abstract
Single-cell RNA sequencing (scRNA-seq) distinguishes cell types, states and lineages within the context of heterogeneous tissues. However, current single-cell data cannot directly link cell clusters with specific phenotypes. Here we present Scissor, a method that identifies cell subpopulations from single-cell data that are associated with a given phenotype. Scissor integrates phenotype-associated bulk expression data and single-cell data by first quantifying the similarity between each single cell and each bulk sample. It then optimizes a regression model on the correlation matrix with the sample phenotype to identify relevant subpopulations. Applied to a lung cancer scRNA-seq dataset, Scissor identified subsets of cells associated with worse survival and with TP53 mutations. In melanoma, Scissor discerned a T cell subpopulation with low PDCD1/CTLA4 and high TCF7 expression associated with an immunotherapy response. Beyond cancer, Scissor was effective in interpreting facioscapulohumeral muscular dystrophy and Alzheimer's disease datasets. Scissor identifies biologically and clinically relevant cell subpopulations from single-cell assays by leveraging phenotype and bulk-omics datasets.
Single-cell RNA sequencing (scRNA-seq) distinguishes cell types, states and lineages within the context of heterogeneous tissues. However, current single-cell data cannot directly link cell clusters with specific phenotypes. Here we present Scissor, a method that identifies cell subpopulations from single-cell data that are associated with a given phenotype. Scissor integrates phenotype-associated bulk expression data and single-cell data by first quantifying the similarity between each single cell and each bulk sample. It then optimizes a regression model on the correlation matrix with the sample phenotype to identify relevant subpopulations. Applied to a lung cancer scRNA-seq dataset, Scissor identified subsets of cells associated with worse survival and with TP53 mutations. In melanoma, Scissor discerned a T cell subpopulation with low PDCD1/CTLA4 and high TCF7 expression associated with an immunotherapy response. Beyond cancer, Scissor was effective in interpreting facioscapulohumeral muscular dystrophy and Alzheimer's disease datasets. Scissor identifies biologically and clinically relevant cell subpopulations from single-cell assays by leveraging phenotype and bulk-omics datasets.
Polesso F, Munks MW, Rott KH, Smart S, Hill AB, Moran AE. PD-1-specific "Blocking" antibodies that deplete PD-1+ T cells present an inconvenient variable in preclinical immunotherapy experiments. Eur J Immunol. 2021 Jun;51(6):1473-1481. doi: 10.1002/eji.202048960. Epub 2021 Mar 23. PMID: 33684223.
Abstract
Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.
Therapeutic antibodies blocking PD-1-/PD-L1 interaction have achieved remarkable clinical success in cancer. In addition to blocking a target molecule, some isotypes of antibodies can activate complement, NK cells or phagocytes, resulting in death of the cell expressing the antibody's target. Human anti-PD-1 therapeutics use antibody isotypes designed to minimize such antibody-dependent lysis. In contrast, anti-PD-1 reagents used in mice are derived from multiple species, with different isotypes, and are not engineered to reduce target cell death: few studies analyze or discuss how antibody species and isotype may impact data interpretation. We demonstrate here that anti-PD-1 therapy to promote activation and proliferation of murine PD-1-expressing CD8 T cells sometimes led instead to a loss of antigen specific cells. This phenomenon was seen in two tumor models and a model of virus infection, and varied with the clone of anti-PD-1 antibody. Additionally, we compared competition among anti-PD-1 clones to find a combination that allows detection of PD-1-expressing cells despite the presence of blocking anti-PD1 antibodies in vivo. These data bring attention to the possibility of unintended target cell depletion with some commonly used anti-mouse PD-1 clones, and should provide a valuable resource for the design and interpretation of anti-PD-1 studies in mice.
Graff JN, Beer TM, Alumkal JJ, Slottke RE, Redmond WL, Thomas GV, Thompson RF, Wood MA, Koguchi Y, Chen Y, Latour E, Bergan RC, Drake CG, Moran AE. A phase II single-arm study of pembrolizumab with enzalutamide in men with metastatic castration-resistant prostate cancer progressing on enzalutamide alone. J Immunother Cancer. 2020 Jul;8(2):e000642. doi: 10.1136/jitc-2020-000642. PMID: 32616555; PMCID: PMC7333874.
Abstract
Background: Checkpoint inhibitors can induce profound anticancer responses, but programmed cell death protein-1 (PD-1) inhibition monotherapy has shown minimal activity in prostate cancer. A published report showed that men with prostate cancer who were resistant to the second-generation androgen receptor inhibitor enzalutamide had increased programmed death-ligand 1 (PD-L1) expression on circulating antigen-presenting cells. We hypothesized that the addition of PD-1 inhibition in these patients could induce a meaningful cancer response.
Methods: We evaluated enzalutamide plus the PD-1 inhibitor pembrolizumab in a single-arm phase II study of 28 men with metastatic castration-resistant prostate cancer (mprogressing on enzalutamide alone. Pembrolizumab 200 mg intravenous was given every 3 weeks for four doses with enzalutamide. The primary endpoint was prostate-specific antigen (PSA) decline of ≥50%. Secondary endpoints were objective response, PSA progression-free survival (PFS), time to subsequent treatment, and time to death. Baseline tumor biopsies were obtained when feasible, and samples were sequenced and evaluated for the expression of PD-L1, microsatellite instability (MSI), mutational and neoepitope burdens.
Results: Five (18%) of 28 patients had a PSA decline of ≥50%. Three (25%) of 12 patients with measurable disease at baseline achieved an objective response. Of the five responders, two continue with PSA and radiographic response after 39.3 and 37.8 months. For the entire cohort, median follow-up was 37 months, and median PSA PFS time was 3.8 months (95% CI: 2.8 to 9.9 months). Time to subsequent treatment was 7.21 months (95% CI: 5.1 to 11.1 months). Median overall survival for all patients was 21.9 months (95% CI: 14.7 to 28 .4 months), versus 41.7 months (95% CI: 22.16 to not reached (NR)) in the responders. Of the three responders with baseline biopsies, one had MSI high disease with mutations consistent with DNA-repair defects. None had detectable PD-L1 expression.
Background: Checkpoint inhibitors can induce profound anticancer responses, but programmed cell death protein-1 (PD-1) inhibition monotherapy has shown minimal activity in prostate cancer. A published report showed that men with prostate cancer who were resistant to the second-generation androgen receptor inhibitor enzalutamide had increased programmed death-ligand 1 (PD-L1) expression on circulating antigen-presenting cells. We hypothesized that the addition of PD-1 inhibition in these patients could induce a meaningful cancer response.
Methods: We evaluated enzalutamide plus the PD-1 inhibitor pembrolizumab in a single-arm phase II study of 28 men with metastatic castration-resistant prostate cancer (mprogressing on enzalutamide alone. Pembrolizumab 200 mg intravenous was given every 3 weeks for four doses with enzalutamide. The primary endpoint was prostate-specific antigen (PSA) decline of ≥50%. Secondary endpoints were objective response, PSA progression-free survival (PFS), time to subsequent treatment, and time to death. Baseline tumor biopsies were obtained when feasible, and samples were sequenced and evaluated for the expression of PD-L1, microsatellite instability (MSI), mutational and neoepitope burdens.
Results: Five (18%) of 28 patients had a PSA decline of ≥50%. Three (25%) of 12 patients with measurable disease at baseline achieved an objective response. Of the five responders, two continue with PSA and radiographic response after 39.3 and 37.8 months. For the entire cohort, median follow-up was 37 months, and median PSA PFS time was 3.8 months (95% CI: 2.8 to 9.9 months). Time to subsequent treatment was 7.21 months (95% CI: 5.1 to 11.1 months). Median overall survival for all patients was 21.9 months (95% CI: 14.7 to 28 .4 months), versus 41.7 months (95% CI: 22.16 to not reached (NR)) in the responders. Of the three responders with baseline biopsies, one had MSI high disease with mutations consistent with DNA-repair defects. None had detectable PD-L1 expression.
Maniar A, Moran AE, Graff JN. The Relationship Between Checkpoint Inhibitors and the Gut Microbiome and Its Application in Prostate Cancer. Oncology (Williston Park). 2020;34(3). Epub 2020/03/27. PubMed PMID: 32212131.
Abstract
Indications for checkpoint inhibitors (CPIs) are growing rapidly within the field of oncology; however, they continue to have heterogeneous outcomes in different cancers. Other than mismatch repair deficiency, there are no consistent tests to determine a tumor's susceptibility. By exploring factors beyond the cancer cell, researchers have learned that the efficacy of CPIs may be governed by a myriad of variable host factors, including the tumor microenvironment (TME) and gut microbiome (GMB). The GMB serves as one of the primary organs of immune defense and has well-established local and systemic effects on the host immune system. Recent investigations suggest that the GMB also affects the TME. This review article discusses the concepts of a TME and a GMB and their effects on responses to CPIs. It also reviews recent research investigating these 3 topics, and how it can be applied to using CPIs in prostate cancer. By highlighting this important pathophysiologic process, we hope to provide insight into a possible explanation for differences in interindividual response to CPIs, discuss a potential method for transferring treatment efficacy between patients, and propose a method for expanding the use of CPIs to prostate cancer.
Indications for checkpoint inhibitors (CPIs) are growing rapidly within the field of oncology; however, they continue to have heterogeneous outcomes in different cancers. Other than mismatch repair deficiency, there are no consistent tests to determine a tumor's susceptibility. By exploring factors beyond the cancer cell, researchers have learned that the efficacy of CPIs may be governed by a myriad of variable host factors, including the tumor microenvironment (TME) and gut microbiome (GMB). The GMB serves as one of the primary organs of immune defense and has well-established local and systemic effects on the host immune system. Recent investigations suggest that the GMB also affects the TME. This review article discusses the concepts of a TME and a GMB and their effects on responses to CPIs. It also reviews recent research investigating these 3 topics, and how it can be applied to using CPIs in prostate cancer. By highlighting this important pathophysiologic process, we hope to provide insight into a possible explanation for differences in interindividual response to CPIs, discuss a potential method for transferring treatment efficacy between patients, and propose a method for expanding the use of CPIs to prostate cancer.
Graff JN, Stein MN, Surana R, Al Rabadi L, Liu E, Fong L, Bailey S, Latour E, Newby TA, Moran AE, Beer TM. Phase II Study of Ipilimumab in Men With Metastatic Prostate Cancer With an Incomplete Response to Androgen Deprivation Therapy. Front Oncol. 2020 Aug 7;10:1381. doi: 10.3389/fonc.2020.01381. PMID: 32850444; PMCID: PMC7426513.
Abstract
Background: Phase 3 studies of immune checkpoint inhibitors have not shown a survival benefit in prostate cancer, but some patients have a profound anticancer response.
Patients and Methods: We evaluated the efficacy of the CTLA-4 targeted agent, ipilimumab, in metastatic prostate cancer patients who had an incomplete biochemical response to initial androgen deprivation therapy (ADT) alone. Ten patients were enrolled, each treated with ipilimumab 10 mg/kg (every 3 weeks for up to 4 doses) with maintenance ipilimumab every 12 weeks for non-progressing patients. The primary endpoint was proportion of patients with an undetectable PSA. The total sample size was 30 patients, but there was an interim analysis planned at 10 for futility. If none of the 10 patients achieved an undetectable PSA, the study would be halted.
Results: The study was halted at the interim analysis as none of the 10 patients achieved the primary endpoint, but 30% of patients demonstrated a >50% reduction in PSA, with one patient achieving a >90% reduction in PSA. Peripheral blood mononuclear cells (PBMC) examined by mass cytometry showed that patients with clinical responses had an increase in effector memory T-cell subsets as well as an increase in T-cell expression of T-bet, suggesting induction of a Th1 response. =
Conclusions: This study provides further evidence that ipilimumab has activity in some patients with prostate cancer and provides further rationale for the development of future studies aimed at identifying a subset of patients with CPRC that are more likely to derive a benefit from treatment with ipilimumab.
Implications for Practice: There is insufficient evidence to use ipilimumab in prostate cancer in routine practice.
Background: Phase 3 studies of immune checkpoint inhibitors have not shown a survival benefit in prostate cancer, but some patients have a profound anticancer response.
Patients and Methods: We evaluated the efficacy of the CTLA-4 targeted agent, ipilimumab, in metastatic prostate cancer patients who had an incomplete biochemical response to initial androgen deprivation therapy (ADT) alone. Ten patients were enrolled, each treated with ipilimumab 10 mg/kg (every 3 weeks for up to 4 doses) with maintenance ipilimumab every 12 weeks for non-progressing patients. The primary endpoint was proportion of patients with an undetectable PSA. The total sample size was 30 patients, but there was an interim analysis planned at 10 for futility. If none of the 10 patients achieved an undetectable PSA, the study would be halted.
Results: The study was halted at the interim analysis as none of the 10 patients achieved the primary endpoint, but 30% of patients demonstrated a >50% reduction in PSA, with one patient achieving a >90% reduction in PSA. Peripheral blood mononuclear cells (PBMC) examined by mass cytometry showed that patients with clinical responses had an increase in effector memory T-cell subsets as well as an increase in T-cell expression of T-bet, suggesting induction of a Th1 response. =
Conclusions: This study provides further evidence that ipilimumab has activity in some patients with prostate cancer and provides further rationale for the development of future studies aimed at identifying a subset of patients with CPRC that are more likely to derive a benefit from treatment with ipilimumab.
Implications for Practice: There is insufficient evidence to use ipilimumab in prostate cancer in routine practice.
Polesso F, Sarker M, Weinberg AD, Murray SE, Moran AE. OX40 Agonist Tumor Immunotherapy Does Not Impact Regulatory T Cell Suppressive Function. J Immunol. 2019 Oct 1;203(7):2011-2019. doi: 10.4049/jimmunol.1900696. Epub 2019 Aug 21. DOI: 10.4049/jimmunol.1900696 PMID: 31434709.
Abstract
OX40 is a costimulatory molecule from the TNFR family. In mice, it is expressed on Foxp3+ regulatory T cells (Tregs) constitutively and on conventional CD4 (Tconv) and CD8 T cells after Ag encounter. OX40 agonists are in clinical development to enhance antitumor immune responses, and one proposed mechanism of action is loss of Treg suppressive function. Studies have postulated that agonist OX40 therapy can impair Treg suppressive function. Using tools developed since the initial studies were published, we evaluated a direct effect of OX40 agonism on Treg function. We conclude that OX40 agonist Abs do not intrinsically impair Treg function but rather enhance Tconv cell IL-2 production, increasing Treg and Tconv cell proliferation. OX40-stimulated Tregs retain suppressive function, but also gain IFN-γ, TNF-α, and granzyme B expression. These data help resolve mechanistic questions regarding OX40 agonist immunotherapy and thus are relevant to developing combination therapies that target distinct T cell functions.
OX40 is a costimulatory molecule from the TNFR family. In mice, it is expressed on Foxp3+ regulatory T cells (Tregs) constitutively and on conventional CD4 (Tconv) and CD8 T cells after Ag encounter. OX40 agonists are in clinical development to enhance antitumor immune responses, and one proposed mechanism of action is loss of Treg suppressive function. Studies have postulated that agonist OX40 therapy can impair Treg suppressive function. Using tools developed since the initial studies were published, we evaluated a direct effect of OX40 agonism on Treg function. We conclude that OX40 agonist Abs do not intrinsically impair Treg function but rather enhance Tconv cell IL-2 production, increasing Treg and Tconv cell proliferation. OX40-stimulated Tregs retain suppressive function, but also gain IFN-γ, TNF-α, and granzyme B expression. These data help resolve mechanistic questions regarding OX40 agonist immunotherapy and thus are relevant to developing combination therapies that target distinct T cell functions.
Polesso F, Weinberg A, Moran AE. Late stage tumor regression after PD-L1 blockade with a concurrent OX40 agonist. Cancer Immunol Res. 2018 Dec 18. pii: canimm.0222.2018. doi: 10.1158/2326-6066.CIR-18-0222. [Epub ahead of print.]
PMID: 30563828.
PMID: 30563828.
Abstract
The protective capability of tumor antigen-specific T cells is regulated by costimulatory and inhibitory signals. Current approaches in cancer immunotherapy seek to restore the function of unresponsive T cells by blocking inhibitory pathways. In contrast, providing exogenous costimulatory signals to T cells also enhances antitumor functionality. By combining these two clinical approaches, we demonstrate the synergy of targeting PD-L1 together with the costimulatory molecule OX40, to enhance antitumor immunity. Concurrently blocking PD-L1 and providing a costimulatory agonist to OX40 increased the presence and functionality of tumor antigen-specific CD8+ T cells with simultaneous enhancement of T-helper type 1 (Th1)-skewed CD4+ T cells. This shift was functionally supported by increased glucose metabolism of antigen-specific CD8+ T cells and the acquisition of granzyme B by regulatory T cells. Together, this mechanism promoted tumor regression of late-stage tumors beyond that achieved by either blockade as monotherapy. These findings indicate that targeting both T-cell intrinsic (OX40) and extrinsic (PD-L1) regulatory molecules increases the bioenergetic potential of T cells, thereby expanding functional and tumor antigen-specific T cells.
The protective capability of tumor antigen-specific T cells is regulated by costimulatory and inhibitory signals. Current approaches in cancer immunotherapy seek to restore the function of unresponsive T cells by blocking inhibitory pathways. In contrast, providing exogenous costimulatory signals to T cells also enhances antitumor functionality. By combining these two clinical approaches, we demonstrate the synergy of targeting PD-L1 together with the costimulatory molecule OX40, to enhance antitumor immunity. Concurrently blocking PD-L1 and providing a costimulatory agonist to OX40 increased the presence and functionality of tumor antigen-specific CD8+ T cells with simultaneous enhancement of T-helper type 1 (Th1)-skewed CD4+ T cells. This shift was functionally supported by increased glucose metabolism of antigen-specific CD8+ T cells and the acquisition of granzyme B by regulatory T cells. Together, this mechanism promoted tumor regression of late-stage tumors beyond that achieved by either blockade as monotherapy. These findings indicate that targeting both T-cell intrinsic (OX40) and extrinsic (PD-L1) regulatory molecules increases the bioenergetic potential of T cells, thereby expanding functional and tumor antigen-specific T cells.
Moran AE, Polesso F, Weinberg AD. Immunotherapy expands and maintains the function of high affinity tumor infiltrating CD8 T cells in situ. (2016) Journal of Immunology. Aug 8 epub. PMID: 27503208.
Abstract
Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.
Cancer cells harbor high-affinity tumor-associated Ags capable of eliciting potent antitumor T cell responses, yet detecting these polyclonal T cells is challenging. Therefore, surrogate markers of T cell activation such as CD69, CD44, and programmed death-1 (PD-1) have been used. We report in this study that in mice, expression of activation markers including PD-1 is insufficient in the tumor microenvironment to identify tumor Ag-specific T cells. Using the Nur77GFP T cell affinity reporter mouse, we highlight that PD-1 expression can be induced independent of TCR ligation within the tumor. Given this, we characterized the utility of the Nur77GFP model system in elucidating mechanisms of action of immunotherapies independent of PD-1 expression. Coexpression of Nur77GFP and OX40 identifies a polyclonal population of high-affinity tumor-associated Ag-specific CD8(+) T cells, which produce more IFN-γ in situ than OX40 negative and doubles in quantity with anti-OX40 and anti-CTLA4 mAb therapy but not with anti-PD-1 or programmed death ligand-1. Moreover, expansion of these high-affinity CD8 T cells prolongs survival of tumor-bearing animals. Upon chronic stimulation in tumors and after adoptive cell therapy, CD8 TCR signaling and Nur77GFP induction is impaired, and tumors progress. However, this can be reversed and overall survival significantly enhanced after adoptive cell therapy with agonist OX40 immunotherapy. Therefore, we propose that OX40 agonist immunotherapy can maintain functional TCR signaling of chronically stimulated tumor-resident CD8 T cells, thereby increasing the frequency of cytotoxic, high-affinity, tumor-associated Ag-specific cells.
Dillon TJ, Takahashi M, Li Y, Tavisala S, Murray SE, Moran AE, Parker DC, Stork PJ. B-Raf is required for positive selection and survival of DP cells, but not for negative selection of SP cells. (2013). International Immunology. April 25(4):259-69.
Abstract
The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.
The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.
Moran AE, Kovacsovics-Bankowski M, Weinberg AD. The TNFRs OX40, 4-1BB, and CD40 as targets for cancer immunotherapy. (2013). Current Opinion in Immunology. April 25(2):230-7.
Abstract
T cell-mediated rejection of tumors requires signals from the T cell receptor and co-stimulatory molecules to license effector functions of tumor-antigen specific T cells. There is also an array of immune suppressive mechanisms within the tumor microenvironment that can suppress anti-tumor immunity. The use of monoclonal antibodies to overcome this suppression and/or enhance tumor-antigen specific T cell responses has shown promise in clinical trials. In particular, targeting co-stimulatory members of the tumor necrosis factor receptor (TNFR) family with agonist Abs enhances T cell function, which has led to encouraging therapeutic results in cancer-bearing hosts. These encouraging data establish TNFRs as important targets for enhancing tumor-specific immune responses in mice and man. This review will focus on agonists that target the TNFRs OX40, 4-1BB, and CD40.
T cell-mediated rejection of tumors requires signals from the T cell receptor and co-stimulatory molecules to license effector functions of tumor-antigen specific T cells. There is also an array of immune suppressive mechanisms within the tumor microenvironment that can suppress anti-tumor immunity. The use of monoclonal antibodies to overcome this suppression and/or enhance tumor-antigen specific T cell responses has shown promise in clinical trials. In particular, targeting co-stimulatory members of the tumor necrosis factor receptor (TNFR) family with agonist Abs enhances T cell function, which has led to encouraging therapeutic results in cancer-bearing hosts. These encouraging data establish TNFRs as important targets for enhancing tumor-specific immune responses in mice and man. This review will focus on agonists that target the TNFRs OX40, 4-1BB, and CD40.
Casey KA, Fraser KA, Schenkel JM, Moran AE, Abt MC, Beura LK, Lucas PJ, Artis D, Wherry EJ, Hogquist K, Vezys V, Masopust D. Antigen-Independent Differentiation and Maintenance of Effector-like Resident Memory T cells in Tissues. (2012). Journal of Immunology. May 15; 188(10): 4866-75.
PMID: 22504644.
PMID: 22504644.
Abstract
Differentiation and maintenance of recirculating effector memory CD8 T cells (TEM) depends on prolonged cognate antigen stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (TRM), which contribute to rapid local protection upon pathogen re-exposure, is unknown. Memory CD8αβ+ T cells within small intestine epithelium are well-characterized examples of TRM and they maintain a long-lived effector-like phenotype that is highly suggestive of persistent antigen stimulation. This study sought to define the sources and requirements for prolonged Ag-stimulation in programming this differentiation state, including local stimulation via cognate or cross-reactive antigens derived from pathogens, microbial flora, or dietary proteins. Contrary to expectations, we found that prolonged cognate Ag-stimulation was dispensable for intestinal TRM ontogeny. In fact, chronic antigenic stimulation skewed differentiation away from the canonical intestinal T cell phenotype. Resident memory signatures, CD69 and CD103, were expressed in many non-lymphoid tissues including intestine, stomach, kidney, reproductive tract, pancreas, brain, heart, and salivary gland, and could be driven by cytokines. Moreover, TGFβ driven CD103 expression was required for TRM maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal TRM differed from classic models of TEM ontogeny, and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.
Differentiation and maintenance of recirculating effector memory CD8 T cells (TEM) depends on prolonged cognate antigen stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (TRM), which contribute to rapid local protection upon pathogen re-exposure, is unknown. Memory CD8αβ+ T cells within small intestine epithelium are well-characterized examples of TRM and they maintain a long-lived effector-like phenotype that is highly suggestive of persistent antigen stimulation. This study sought to define the sources and requirements for prolonged Ag-stimulation in programming this differentiation state, including local stimulation via cognate or cross-reactive antigens derived from pathogens, microbial flora, or dietary proteins. Contrary to expectations, we found that prolonged cognate Ag-stimulation was dispensable for intestinal TRM ontogeny. In fact, chronic antigenic stimulation skewed differentiation away from the canonical intestinal T cell phenotype. Resident memory signatures, CD69 and CD103, were expressed in many non-lymphoid tissues including intestine, stomach, kidney, reproductive tract, pancreas, brain, heart, and salivary gland, and could be driven by cytokines. Moreover, TGFβ driven CD103 expression was required for TRM maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal TRM differed from classic models of TEM ontogeny, and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.
Moran AE, Hogquist KA. T cell receptor affinity in thymic development. (2012). Immunology. April; 135(4):261-7. PMID: 22182461.
Abstract
Understanding the thymic processes that support the generation of functionally competent and self-tolerant lymphocytes requires dissection of the T-cell receptor (TCR) response to ligands of different affinities. In spatially segregated regions of the thymus, with unique expression of proteases and cytokines, TCR affinity guides a number of cell fate decisions. Yet affinity alone does not explain the selection paradox. Increasing evidence suggests that the 'altered peptide' model of the 1980s together with the affinity model might best explain how the thymus supports conventional and regulatory T-cell development. Development of new tools to study the strength of TCR signals perceived by T cells, novel regulatory T-cell transgenic mice, and tetramer enrichment strategies have provided an insight into the nature of TCR signals perceived during thymocyte development. These topics are discussed and support for the prevailing hypotheses is presented.
Understanding the thymic processes that support the generation of functionally competent and self-tolerant lymphocytes requires dissection of the T-cell receptor (TCR) response to ligands of different affinities. In spatially segregated regions of the thymus, with unique expression of proteases and cytokines, TCR affinity guides a number of cell fate decisions. Yet affinity alone does not explain the selection paradox. Increasing evidence suggests that the 'altered peptide' model of the 1980s together with the affinity model might best explain how the thymus supports conventional and regulatory T-cell development. Development of new tools to study the strength of TCR signals perceived by T cells, novel regulatory T-cell transgenic mice, and tetramer enrichment strategies have provided an insight into the nature of TCR signals perceived during thymocyte development. These topics are discussed and support for the prevailing hypotheses is presented.
Moran AE, Holzapfel KL, Xing Y, Cunningham NR, Maltzman JS, Punt J, Hogquist KA. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. (2011). Journal of Experimental Medicine. May 23, Epub. PMCID: 3173240.
Abstract
The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (Treg cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although Treg cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that Treg cell progenitors compete for recognition of rare stimulatory TCR self-ligands.
The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (Treg cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although Treg cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that Treg cell progenitors compete for recognition of rare stimulatory TCR self-ligands.
Hogquist KA & Moran AE. Treg cells meet their limit. Nature Immunology. 2009 June 6 (10):565-6.
Abstract
T cell antigen receptor (TCR)-transgenic models have been enormously influential in studies of T cell development in the thymus, particularly in terms of positive and negative selection. New transgenic mice produced with TCR genes cloned from regulatory T cells show that TCR specificity does 'instruct' regulatory T cell fate, within limits.
T cell antigen receptor (TCR)-transgenic models have been enormously influential in studies of T cell development in the thymus, particularly in terms of positive and negative selection. New transgenic mice produced with TCR genes cloned from regulatory T cells show that TCR specificity does 'instruct' regulatory T cell fate, within limits.
Wirth, LJ, Krane, JF, Li Y, Othus, M, Moran AE, Dorfman DM, Norris CM, Goguen L, Posner MR, Haddad RI, Bertagnolli MM. A Pilot Surrogate Endpoint Biomarker Study of Celecoxib in Oral Premalignant Lesions. Cancer Prevention Research. 2008 Oct 1; (5):339-48.
Abstract
This study evaluated changes in prostaglandin E(2) (PGE(2)) levels and related biomarkers in oral premalignant lesions (OPL) in response to celecoxib treatment. Twenty-two subjects were enrolled and treated with celecoxib. Pretreatment and 12-week biopsies were done. Subjects whose biopsy showed >or=30% decrease in PGE(2) remained on celecoxib for a total of 12 months when repeat biopsy was done. Biopsies were examined to assess degree of dysplasia, DNA ploidy, and immunohistochemical expression of BCL2, pAKT-Ser473, Ki-67, and CD31 (microvessel density). In 18 paired biopsies available at baseline and 12 weeks, mean normalized PGE(2) levels decreased by 38% (P = 0.002). After 12 months, PGE(2) decreased by 31% (P = 0.340). Twelve biopsies (67%; P = 0.0129) showed improvement in degree of dysplasia after 12 weeks, and 8 of 11 biopsies (73%; P = 0.0703) continued to show an improvement in the degree of dysplasia after 12 months. Trends suggested down-modulation of cyclooxygenase-2 and Ki-67 in some tissues, increased pAKT-Ser473 expression, and an inverse relationship between PGE(2) and BCL2 expression. This study documents the feasibility of measuring potential surrogate endpoint biomarkers of chemopreventive agent response in OPLs. Treatment with celecoxib in subjects with OPLs favorably modulates the primary mediator of cyclooxygenase-2 activity, PGE(2), after 12 weeks.
This study evaluated changes in prostaglandin E(2) (PGE(2)) levels and related biomarkers in oral premalignant lesions (OPL) in response to celecoxib treatment. Twenty-two subjects were enrolled and treated with celecoxib. Pretreatment and 12-week biopsies were done. Subjects whose biopsy showed >or=30% decrease in PGE(2) remained on celecoxib for a total of 12 months when repeat biopsy was done. Biopsies were examined to assess degree of dysplasia, DNA ploidy, and immunohistochemical expression of BCL2, pAKT-Ser473, Ki-67, and CD31 (microvessel density). In 18 paired biopsies available at baseline and 12 weeks, mean normalized PGE(2) levels decreased by 38% (P = 0.002). After 12 months, PGE(2) decreased by 31% (P = 0.340). Twelve biopsies (67%; P = 0.0129) showed improvement in degree of dysplasia after 12 weeks, and 8 of 11 biopsies (73%; P = 0.0703) continued to show an improvement in the degree of dysplasia after 12 months. Trends suggested down-modulation of cyclooxygenase-2 and Ki-67 in some tissues, increased pAKT-Ser473 expression, and an inverse relationship between PGE(2) and BCL2 expression. This study documents the feasibility of measuring potential surrogate endpoint biomarkers of chemopreventive agent response in OPLs. Treatment with celecoxib in subjects with OPLs favorably modulates the primary mediator of cyclooxygenase-2 activity, PGE(2), after 12 weeks.
*Moran AE, *Carothers AM, Cho NL, Redston M, Bertagnolli MM. Changes in antitumor response in C57BL/6J-Min/+ mice during long-term administration of a selective cyclooxegenase-2 inhibitor. Cancer Research. 2006 Sep 5; 114(10):1028-35 *equal contribution.
Abstract
Selective cyclooxygenase-2 (COX-2) inhibitors are widely prescribed for severe arthritis and are currently under study in human chemoprevention trials. Recently, long-term use of these agents has come under scrutiny due to reports of treatment-associated cardiovascular toxicity. On short-term administration, the selective COX-2 inhibitor celecoxib inhibits adenoma growth in animal tumor models, including the C57BL/6J-Min/+ (Min/+) mouse. With uninterrupted long-term celecoxib administration, intestinal tumors in Min/+ mice initially regressed and then recurred to levels comparable with untreated controls. Celecoxib treatment initially suppressed COX-2 and prostaglandin E2 (PGE2) expression, but long-term use produced significantly higher levels of these molecules and reactivated PGE2-associated growth factor signaling pathways in tumor and normal tissues. These results indicate that COX-2 is an important chemoprevention target and that inhibition of this enzyme alters a paracrine enterocyte regulatory pathway. Chronic uninterrupted celecoxib treatment, however, induces untoward effects that enhance early progression events in intestinal tumorigenesis and may contribute to treatment toxicity.
Selective cyclooxygenase-2 (COX-2) inhibitors are widely prescribed for severe arthritis and are currently under study in human chemoprevention trials. Recently, long-term use of these agents has come under scrutiny due to reports of treatment-associated cardiovascular toxicity. On short-term administration, the selective COX-2 inhibitor celecoxib inhibits adenoma growth in animal tumor models, including the C57BL/6J-Min/+ (Min/+) mouse. With uninterrupted long-term celecoxib administration, intestinal tumors in Min/+ mice initially regressed and then recurred to levels comparable with untreated controls. Celecoxib treatment initially suppressed COX-2 and prostaglandin E2 (PGE2) expression, but long-term use produced significantly higher levels of these molecules and reactivated PGE2-associated growth factor signaling pathways in tumor and normal tissues. These results indicate that COX-2 is an important chemoprevention target and that inhibition of this enzyme alters a paracrine enterocyte regulatory pathway. Chronic uninterrupted celecoxib treatment, however, induces untoward effects that enhance early progression events in intestinal tumorigenesis and may contribute to treatment toxicity.
Carothers AM, Javid SH, Moran AE, Hunt DH, Redston M, Bertagnolli MM. Deficient E-cadherin adhesion in C57BL/6J-Min/+ mice is associated with increased tyrosine kinase activity and RhoA-dependent actomysin contractility. Experimental Cell Research 2006 Feb 15; 312(4):387-400.
Abstract
The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.
The Min/+ mouse is a model for APC-dependent colorectal cancer (CRC). We showed that tumorigenesis in this animal was associated with decreased E-cadherin adhesion and increased epidermal growth factor receptor (Egfr) activity in the non-tumor intestinal mucosa. Here, we tested whether these abnormalities correlated with changes in the actin cytoskeleton due to increased Rho-ROCK signaling. We treated Apc+/+ (WT) littermate small intestine with EGTA, an inhibitor of E-cadherin, and with LPA, an RhoA activator; both induced effects on adhesion and kinase activity that mimicked the Min/+ phenotype. GTP-bound Rho was increased in Min/+ enterocytes relative to WT. Since RhoA activity is associated with actomyosin contractility, markers of this signaling cascade were assessed including phosphorylated myosin light chain (MLC), cofilin, Pyk2, Src, and MAPK kinases. The increased actomyosin contractility characterizing Min/+ intestinal tissue was suppressed by the ROCK inhibitor, Y27632, but was inducible in the WT by EGTA or LPA. Finally, ultrastructural imaging revealed changes consistent with actomyosin contractility in Min/+ enterocytes. Thus, the positive regulation of E-cadherin adhesion provided by Apc+ in vivo allows proper negative regulation of Egfr, Src, Pyk2, and MAPK, as well as RhoA activities.
Moran AE, Carothers AM, Weyant MJ, Redston M, Bertagnolli MM. Carnosol inhibits beta-catenin tyrosine phosphorylation and prevents adenoma formation in the C57BL/6J/Min/+ (Min/+) mouse. Cancer Research. 2005 Feb 1; 65(3):1097-104.
Abstract
Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.
Carnosol, a constituent of the herb, rosemary, has shown beneficial medicinal and antitumor effects. Using the C57BL/6J/Min/+ (Min/+) mouse, a model of colonic tumorigenesis, we found that dietary administration of 0.1% carnosol decreased intestinal tumor multiplicity by 46%. Previous studies showed that tumor formation in the Min/+ mouse was associated with alterations in the adherens junctions, including an increased expression of tyrosine-phosphorylated beta-catenin, dissociation of beta-catenin from E-cadherin, and strongly reduced amounts of E-cadherin located at lateral plasma membranes of histologically normal enterocytes. Here, we confirm these findings and show that treatment of Min/+ intestinal tissue with carnosol restored both E-cadherin and beta-catenin to these enterocyte membranes, yielding a phenotype similar to that of the Apc(+/+) wild-type (WT) littermate. Moreover, treatment of WT intestine with the phosphatase inhibitor, pervanadate, removed E-cadherin and beta-catenin from the lateral membranes of enterocytes, mimicking the appearance of the Min/+ tissue. Pretreatment of WT tissue with carnosol inhibited the pervanadate-inducible expression of tyrosine-phosphorylated beta-catenin. Thus, the Apc(Min) allele produces adhesion defects that involve up-regulated expression of tyrosine-phosphorylated proteins, including beta-catenin. Moreover, these data suggest that carnosol prevents Apc-associated intestinal tumorigenesis, potentially via its ability to enhance E-cadherin-mediated adhesion and suppress beta-catenin tyrosine phosphorylation.
Javid SH, Moran AE, Carothers AM, Redston M, Bertagnolli MM. Modulation of tumor formation and intestinal cell migration by estrogens in the Apc(Min/+) mouse model of colorectal tumorigenesis. Carcinogenesis. 2005 Mar; 26(3):587-95.
Abstract
Epidemiological studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone genistein and coumestrol, are used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the C57BL/6J-Min/+ (Min/+) mouse, an animal model of adenomatous polyposis coli (APC)-associated CRC. Replacement of estradiol (E(2)) in ovariectomized Min/+ mice reduced tumor number to baseline and up-regulated the expression of estrogen receptor beta (ERbeta). We hypothesized that the phytoestrogens genistein and coumestrol would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice. Min/+ and Apc(+/+) (WT) mice were ovariectomized and assigned to either a control diet or treatment with E(2), genistein or coumestrol. Treatment of ovariectomized Min/+ (Min/+ OX) mice with genistein resulted in a non-significant reduction in tumor number. Min/+ OX mice treated with coumestrol had significantly fewer tumors than untreated Min/+ OX controls and the same number of tumors as non-ovariectomized Min/+ mice. Bromodeoxyuridine migration assays also demonstrated that treatment with E(2) or coumestrol improved enterocyte migration rate. Immunoprecipitation and immunohistochemistry analyses showed that impaired association of the adherens junction proteins E-cadherin and beta-catenin in Min/+ mice was improved by treatment with either E(2) or coumestrol. Immunoblot analyses also showed that expression of ERbeta was elevated in enterocytes of Min/+ OX mice treated with E(2) or coumestrol as compared with those of untreated Min/+ OX mice. In conclusion, both coumestrol and E(2) prevent intestinal tumorigenesis and ameliorate enterocyte migration and intercellular adhesion in the Apc(Min/+) mouse model of CRC.
Epidemiological studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone genistein and coumestrol, are used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the C57BL/6J-Min/+ (Min/+) mouse, an animal model of adenomatous polyposis coli (APC)-associated CRC. Replacement of estradiol (E(2)) in ovariectomized Min/+ mice reduced tumor number to baseline and up-regulated the expression of estrogen receptor beta (ERbeta). We hypothesized that the phytoestrogens genistein and coumestrol would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice. Min/+ and Apc(+/+) (WT) mice were ovariectomized and assigned to either a control diet or treatment with E(2), genistein or coumestrol. Treatment of ovariectomized Min/+ (Min/+ OX) mice with genistein resulted in a non-significant reduction in tumor number. Min/+ OX mice treated with coumestrol had significantly fewer tumors than untreated Min/+ OX controls and the same number of tumors as non-ovariectomized Min/+ mice. Bromodeoxyuridine migration assays also demonstrated that treatment with E(2) or coumestrol improved enterocyte migration rate. Immunoprecipitation and immunohistochemistry analyses showed that impaired association of the adherens junction proteins E-cadherin and beta-catenin in Min/+ mice was improved by treatment with either E(2) or coumestrol. Immunoblot analyses also showed that expression of ERbeta was elevated in enterocytes of Min/+ OX mice treated with E(2) or coumestrol as compared with those of untreated Min/+ OX mice. In conclusion, both coumestrol and E(2) prevent intestinal tumorigenesis and ameliorate enterocyte migration and intercellular adhesion in the Apc(Min/+) mouse model of CRC.
Moran AE, Hunt DH, Javid SH, Redston M, Carothers AM, Bertagnolli MM. Apc deficiency is associated with increased Egfr activity in the intestinal enterocytes and adenomas of C57BL/6J-Min/+ mice. Journal of Biological Chemistry. 2004 Oct 8; 279(41):43261-72.
Abstract
Overexpression of the epidermal growth factor receptor (EGFR) and its increased tyrosine kinase activity are implicated in colorectal cancer (CRC) development and malignant progression. The C57BL/6J-Min/+ (Min/+) mouse is a model for CRC and develops numerous intestinal adenomas. We analyzed the normal mucosa of Min/+ and Apc+/+ (WT) littermate mice together with Apc-null adenomas to gain insight into the roles of Egfr in these intestinal tissues. Protein analyses showed that Egfr activity was highest in the tumors, and also up-regulated in Min/+ relative to WT enterocytes. Expression of ubiquitylated Egfr (Egfr-Ub) was increased in Min/+ enterocytes and tumors. Tumors exhibited increased association of Egfr with clathrin heavy chain (CHC), Gab1, and p85alpha, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and tumors also overexpressed c-Src, PDK1, and Akt. Immunohistochemistry for Akt-p-Ser473 revealed a low level of this active kinase in Min/+ and WT enterocytes and its strong presence in tumors. Prostaglandin E2 (PGE2) is a product of cyclooxygenase-2 (Cox-2) activity that is up-regulated in Min/+ tumors and transactivates Egfr. PGE2 expression was significantly higher in untreated Min/+ tumors and reduced by treatment with the Cox-2 inhibitor, celecoxib. Dietary administration of this NSAID also inhibited Egfr activity in tumors. Increased activation of the EGFR-PI3K-Akt signaling pathway in tumors relative to Apc+/+ and ApcMin/+ enterocytes provides potential opportunities for therapeutic interventions to differentially suppress tumor formation, promotion, progression, and/or recurrence.
Overexpression of the epidermal growth factor receptor (EGFR) and its increased tyrosine kinase activity are implicated in colorectal cancer (CRC) development and malignant progression. The C57BL/6J-Min/+ (Min/+) mouse is a model for CRC and develops numerous intestinal adenomas. We analyzed the normal mucosa of Min/+ and Apc+/+ (WT) littermate mice together with Apc-null adenomas to gain insight into the roles of Egfr in these intestinal tissues. Protein analyses showed that Egfr activity was highest in the tumors, and also up-regulated in Min/+ relative to WT enterocytes. Expression of ubiquitylated Egfr (Egfr-Ub) was increased in Min/+ enterocytes and tumors. Tumors exhibited increased association of Egfr with clathrin heavy chain (CHC), Gab1, and p85alpha, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and tumors also overexpressed c-Src, PDK1, and Akt. Immunohistochemistry for Akt-p-Ser473 revealed a low level of this active kinase in Min/+ and WT enterocytes and its strong presence in tumors. Prostaglandin E2 (PGE2) is a product of cyclooxygenase-2 (Cox-2) activity that is up-regulated in Min/+ tumors and transactivates Egfr. PGE2 expression was significantly higher in untreated Min/+ tumors and reduced by treatment with the Cox-2 inhibitor, celecoxib. Dietary administration of this NSAID also inhibited Egfr activity in tumors. Increased activation of the EGFR-PI3K-Akt signaling pathway in tumors relative to Apc+/+ and ApcMin/+ enterocytes provides potential opportunities for therapeutic interventions to differentially suppress tumor formation, promotion, progression, and/or recurrence.
Javid SA; Moran AE; Carothers AM, and Bertagnolli MM. Phytoestrogen-mediated suppression of Apc-associated intestinal tumorigenesis. Journal of Surgical Research, Oct. 2003.
Introduction
Epidemiologic studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone, genistein, and coumestrol, are plant-derived estrogenic compounds used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the ApcMin/+ (Min/+) mouse, an animal model of APC-associated CRC. Replacement of estradiol (E2) in ovariectomized Min/+ mice reduced tumor number to baseline and upregulated the expression of estrogen receptor beta (ERβ). We hypothesized that the phytoestrogens, genistein and coumestrol, would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice.
Epidemiologic studies suggest that post-menopausal hormone replacement therapy (HRT) reduces colorectal cancer (CRC) incidence. Phytoestrogens, including the soy isoflavone, genistein, and coumestrol, are plant-derived estrogenic compounds used by many women as alternatives to HRT. Previous studies showed that ovariectomy induced a 77% increase in intestinal adenoma number in the ApcMin/+ (Min/+) mouse, an animal model of APC-associated CRC. Replacement of estradiol (E2) in ovariectomized Min/+ mice reduced tumor number to baseline and upregulated the expression of estrogen receptor beta (ERβ). We hypothesized that the phytoestrogens, genistein and coumestrol, would inhibit intestinal tumorigenesis in ovariectomized Min/+ mice.