A degron system targeting endogenous PD-1 inhibits the growth of tumor cells in mice.
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ABSTRACT:
SUBMITTER: Naruse C
PROVIDER: S-EPMC9204894 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Membrane-bound proteases have recently emerged as critical mediators of tumorigenesis, angiogenesis, and metastasis. However, the mechanisms by which they regulate these processes remain unknown. As the cell surface serine protease fibroblast activation protein (FAP) is selectively expressed on tumor-associated fibroblasts and pericytes in epithelial tumors, we set out to investigate the role of FAP in mouse models of epithelial-derived solid tumors. In this study, we demonstrate that genetic deletion and pharmacologic inhibition of FAP inhibited tumor growth in both an endogenous mouse model of lung cancer driven by the K-rasG12D mutant and a mouse model of colon cancer, in which CT26 mouse colon cancer cells were transplanted into immune competent syngeneic mice. Interestingly, growth of only the K-rasG12D-driven lung tumors was also attenuated by inhibition of the closely related protease dipeptidyl peptidase IV (DPPIV). Our results indicate that FAP depletion inhibits tumor cell proliferation indirectly, increases accumulation of collagen, decreases myofibroblast content, and decreases blood vessel density in tumors. These data provide proof of principle that targeting stromal cell-mediated modifications of the tumor microenvironment may be an effective approach to treating epithelial-derived solid tumors.
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Project description:Activation of the programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/PD-L1) signaling axis plays important roles in intrinsic or acquired resistance to human epidermal growth factor receptor 2 (HER2)-directed therapies in the clinic. Therefore, therapies simultaneously targeting both HER2 and PD-1/PD-L1 signaling pathways are of great significance. Here, aiming to direct the anti-PD-L1 responses toward HER2-expressing tumor cells, we constructed a humanized bispecific IgG1 subclass antibody targeting both HER2 and PD-L1 (HER2/PD-L1; BsAb), which displayed satisfactory purity, thermostability, and serum stability. We found that BsAb showed enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. In the late phase of peripheral blood mononuclear cell (PBMC)-humanized HER2+ tumor xenograft models, BsAb showed superior therapeutic efficacies as compared with monoclonal antibodies (mAbs) or combination treatment strategies. In cynomolgus monkeys, BsAb showed favorable pharmacokinetics and toxicity profiles when administered at a 10 mg/kg dosage. Thus, HER2/PD-L1 BsAb was demonstrated as a potentially effective option for managing HER2+ and trastuzumab-resistant tumors in the clinic. We propose that the enhanced antitumor activities of BsAb in vivo may be due to direct inhibition of HER2 signaling or activation of T cells.
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Project description:BackgroundChimeric antigen receptor T cells (CAR-T cells) therapy has been well recognized for treating B cell-derived malignancy. However, the efficacy of CAR-T cells against solid tumors remains dissatisfactory, partially due to the heterogeneity of solid tumors and T cell exhaustion in tumor microenvironment. PD-L1 is up-regulated in multiple solid tumors, resulting in T cell exhaustion upon binding to its receptor PD-1.MethodsHere, we designed a dominant-negative form of PD-1, dPD1z, a vector containing the extracellular and transmembrane regions of human PD-1, and a CAR vector against PD-L1, CARPD-L1z, a vector employs a high-affinity single-chain variable fragment (scFv) against human PD-L1. These two vectors shared the same intracellular structure, including 4-1BB and TLR2 co-stimulatory domains, and the CD3ζ signaling domain.ResultsdPD1z T and CARPD-L1z T cells efficiently lysed PD-L1+ tumor cells and had enhanced cytokine secretion in vitro and suppressed the growth of non-small cell lung cancer (NSCLC), gastric cancer and hepatoma carcinoma in patient-derived xenograft (PDX). However, the combination of anti-mesothelin CAR-T cells (CARMSLNz T) with dPD1z T or CARPD-L1z T cells did not repress tumor growth synergistically in PDX, as CARMSLNz T cells upregulated PD-L1 expression upon activation and were subsequently attacked by dPD1z T or CARPD-L1z T cells.ConclusionsIn conclusion, we demonstrate CAR-T cells targeting PD-L1 were effective for suppressing the growth of multiple types of solid tumors in PDX models though their safety needs to be carefully examined.
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Project description:CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature.
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Project description:Genetically engineered T cells expressing a T-cell receptor (TCR) are powerful tools for cancer treatment and have shown significant clinical effects in sarcoma patients. However, mismatch of the introduced TCR α/β chains with endogenous TCR may impair the expression of transduced TCR, resulting in an insufficient antitumor capacity of modified T cells. Here, we report the development of immunotherapy using human lymphocytes transduced with a codon-optimized melanoma-associated antigen (MAGE)-A4 and HLA-A*2402-restricted TCR, which specifically downregulate endogenous TCR by small interfering RNA (si-TCR). We evaluated the efficacy of this immunotherapy in both NOD-SCID mice and uterine leiomyosarcoma patients. Our results revealed that transduced human lymphocytes exhibited high surface expression of the introduced tumor-specific TCR, enhanced cytotoxic activity against antigen-expressing tumor cells, and increased interferon-γ production by specific MAGE-A4 peptide stimulation. Retarded tumor growth was also observed in NOD-SCID mice inoculated with human tumor cell lines expressing both MAGE-A4 and HLA-A*2402. Furthermore, we report the successful management of a case of uterine leiomyosarcoma treated with MAGE-A4 si-TCR/HLA-A*2402 gene-modified T cells. Our results indicate that the TCR-modified T cell therapy is a promising novel strategy for cancer treatment.
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Project description:Inhalation therapy using small-interfering RNA (siRNA) is a potentially effective therapeutic strategy for lung cancer because of its high gene-silencing effects and sequence specificity. Previous studies reported that intratracheal administration of siRNA using pressurized metered dose inhalers or nebulizers could suppress tumor growth in murine lung metastatic models. Although dry powder inhalers are promising devices due to their low cost, good portability, and preservability, the anti-tumor effects of siRNA dry powder have not been elucidated. To evaluate the gene-silencing and anti-tumor effects of intratracheally delivered siRNA dry powder, vascular endothelial growth factor-specific siRNA (VEGF-siRNA) dry powder was administered intratracheally to mice with metastatic lung tumors consisting of B16F10 melanoma cells or Lewis lung carcinoma cells. A single intratracheal administration of VEGF-siRNA dry powder reduced VEGF levels in both bronchoalveolar lavage fluid and lung tumor tissue. Furthermore, repeated intratracheal administration of VEGF-siRNA dry powder suppressed the number of visible metastatic foci on the lung surface and tumor area in lung tissues. Taken together, intratracheal administration of siRNA dry powder could be a novel therapeutic strategy for lung cancer through the suppression of specific genes expressed in lung tumor tissue.
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Project description:The suppressive function of T-regulatory cells (Tregs) can have a detrimental effect on immune responses against tumor cells. Within the Treg cells subset, a new non-classical population has been reported, which expresses high levels of CD49b molecule and, depending on their activation status, can also express the canonical Tregs transcription factor Foxp3. In this report, we sought to characterize Tregs subsets in a murine melanoma model and disrupt the CD49b/CD29 axis by administering an anti-CD29 antibody in tumor-bearing mice. Our data shows that whereas in the draining lymph nodes, the Tr1 cells subset composes <5% of CD4+ T cells, in the tumor, they reach ∼30% of CD4+ T cells. Furthermore, Tr1 cells share the expression of suppressive molecules, such as Nrp-1, PD-1, and CD73, which are highly expressed on Tr1 cells found in tumor-infiltrating leukocytes (TILs). Regardless of the phenotypic similarities with cTreg cells, Tr1 cells display a low proliferative activity, as shown in the kinetics and the incorporation of 5-bromodeoxyuridine (BrdU) experiments. With the intent to impact on Tr1 cells, we administered anti-CD29 antibody into tumor mice, observing that the treatment effectively inhibits tumor growth. This effect is at least mediated by the enrichment of pro-inflammatory T cells, including IFN-γ+ cTreg and IFN-γ+ Tr1 cells (with reduced expression of IL-10), plus Th1 and Tc cells. In this study, we present Tr1 cell characterization in tumor-bearing animals and introduce CD29 as a target for tumor therapy, supported by a meta-analysis indicating that CD29 is present in human biopsies.
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Project description:Structural alterations of collagen impact signaling that helps control tumor progression and the responses to therapeutic intervention. Integrins represent a class of receptors that include members that mediate collagen signaling. However, a strategy of directly targeting integrins to control tumor growth has demonstrated limited activity in the clinical setting. New molecular understanding of integrins have revealed that these receptors can regulate both pro‑ and anti‑tumorigenic functions in a cell type‑dependent manner. Therefore, designing strategies that block pro‑tumorigenic signaling, without impeding anti‑tumorigenic functions, may lead to development of more effective therapies. In the present study, evidence was provided for a novel signaling cascade in which β3‑integrin‑mediated binding to a secreted RGDKGE‑containing collagen fragment stimulates an autocrine‑like signaling pathway that differentially governs the activity of both YAP and (protein kinase‑A) PKA, ultimately leading to alterations in the levels of immune checkpoint molecule PD‑L1 by a proteasome dependent mechanism. Selectively targeting this collagen fragment, reduced nuclear YAP levels, and enhanced PKA and proteasome activity, while also exhibiting significant antitumor activity in vivo. The present findings not only provided new mechanistic insight into a previously unknown autocrine‑like signaling pathway that may provide tumor cells with the ability to regulate PD‑L1, but our findings may also help in the development of more effective strategies to control pro‑tumorigenic β3‑integrin signaling without disrupting its tumor suppressive functions in other cellular compartments.
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Project description:BACKGROUND:We previously demonstrated that knockdown of delta-5-desaturase via siRNA transfection together with dihomo-γ-linolenic acid supplementation inhibited colon cancer cell growth and migration, by promoting the production of the anti-cancer byproduct 8-hydroxyoctanoic acid from Cyclooxygenase-2-catalyzed dihomo-γ-linolenic acid peroxidation. Here, we extend our study to investigate the effects of delta-5-desaturase-knockdown and the resulting intensified dihomo-γ-linolenic acid peroxidation in xenograft tumor mice model. METHODS:Four-week old nude mice bearing the human colon cancer cell HCA-7/C29 vs. its delta-5-desaturase knockdown analog (via shRNA transfection) were subject to 4-week treatments of: vehicle control, dihomo-γ-linolenic acid supplementation, 5-Fluorouracil, and combination of dihomo-γ-linolenic acid and 5-Fluorouracil. Tumor growth was monitored during the treatment. At the endpoint, the mice were euthanized and the tumor tissues were collected for further mechanism analysis. RESULTS:Delta-5-desaturase knockdown (shRNA) together with dihomo-γ-linolenic acid supplementation increased 8-hydroxyoctanoic acid production to a threshold level in xenograft tumors, which consequently induced p53-dependent apoptosis and reduced tumors significantly. The promoted 8-hydroxyoctanoic acid formation was also found to suppress the tumors' metastatic potential via regulating MMP-2 and E-cadherin expressions. In addition, our in vivo data showed that delta-5-desaturase knockdown along with dihomo-γ-linolenic acid supplementation resulted in anti-tumor effects comparable to those of 5-Fluorouracil. CONCLUSIONS:We have demonstrated that our paradigm-shifting strategy of knocking down delta-5-desaturase and taking advantage of overexpressed Cyclooxygenase-2 in tumor cells can be used for colon cancer suppression. Our research outcome will lead us to develop a better and safer anti-cancer therapy for patients.
| S-EPMC6299961 | biostudies-literature
Project description:BackgroundImmune checkpoint inhibitor therapy has revolutionized the clinical management of a diverse range of cancer types, including advanced cutaneous melanoma. While immunotherapy targeting the PD-1/PD-L1 system has become standard of care, overall response rates remain unsatisfactory for most patients and there are no approved small molecule inhibitors of the PD-1/PD-L1 system. Flubendazole (FLU) is an anthelmintic that has been used to treat worm infections in humans and animals for decades.MethodsHere we tested the anti-cancer activity of systemically delivered FLU with suppression of PD-1 in immunocompetent mice.ResultsIn C57BL/6J mice bearing subcutaneous B16F10 melanoma, FLU reduced both tumor growth and PD-1 protein levels without affecting levels of PD-L1. FLU's suppression of PD-1 was accompanied by increased CD3+ T cell infiltration. Western blotting with extracts from human Jurkat T cells showed that FLU inhibited PD-1 protein expression, findings confirmed by flow cytometry. To gain mechanistic insights on FLU's ability to suppress PD-1 protein levels, we performed bulk RNA sequencing on extracts of Jurkat T cells exposed to the benzimidazole for 4 h. From a pool of 14,475 genes there were 1218 differentially-expressed genes; 687 with increased expression and 531 with decreased expression. Among the genes induced by FLU was the AP-1 family member, JUN and surprisingly, pdcd1. KEGG pathway analysis showed FLU up-regulated genes over-represented in multiple pathways (p < 0.01), the top hit being amoebiasis. FLU also affected the expression of genes in cancer-associated pathways, both through down-regulation and up-regulation. Gene set enrichment analysis revealed a large number of immunological signature gene sets correlated with FLU treatment, including gene sets associated with T cell differentiation, proliferation and function. The AP-1 inhibitor T5224 rescued PD-1 protein expression from inhibition by FLU.ConclusionThis study is the first to show that FLU can inhibit melanoma growth with PD-1 suppression in immunocompetent mice.
| S-EPMC10349441 | biostudies-literature