Tumor cell‐intrinsic programmed death‐ligand 1 (PD‐L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. ...We show here that BC cell‐intrinsic PD‐L1 signals in mouse MB49 and human RT4, UM‐UC3, and UM‐UC‐14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α‐PD‐L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell‐intrinsic PD‐L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune‐independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell‐intrinsic PD‐L1 signals also promoted basal and stress‐induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell‐intrinsic PD‐L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis‐platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell‐intrinsic PD‐L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof‐of‐concept, we showed that the autophagy inhibitor chloroquine improved cis‐platinum treatment efficacy in vivo, with greater efficacy in PD‐L1 null versus PD‐L1‐replete BC.
We provide evidence that tumor‐intrinsic PD‐L1 mediates pathologic signals in human and mouse bladder cancers, independent of molecular subtype. Signals include control of proliferation, mTORC1, autophagy, and chemotherapy resistance. We show that countering the pathologic cell‐intrinsic PD‐L1 signals improves chemotherapy response in vivo of mice bearing bladder cancer tumors.
Thyroid cancer affects about one percent of the population, and has seen rising incidence in recent years. Follicular thyroid cancer (FTC) comprises 10-15% of all thyroid cancers. Although FTC is ...often localized, it can behave aggressively with hematogenous metastasis, leading to an increased risk of cancer death. We previously described a mouse model for FTC caused by tissue-specific ablation of the Protein Kinase A (PKA) regulatory subunit
, either by itself or in combination with knockout of
. Loss of
causes enhanced activity of PKA, whereas ablation of
causes activation of Akt signaling. At the molecular level, these genetic manipulations caused activation of mTOR signaling, which was also observed in human FTC cases. To understand the mechanism by which PKA activates mTOR, we began by studying intracellular kinases known to modulate mTOR function. Although AMP-activated kinase (AMPK) has been characterized as a negative regulator of mTOR activity, our tumor model exhibited activation of both AMPK and mTOR. To understand the mechanism by which AMPK was turned on, we next studied kinases known to cause its phosphorylation. In this paper, we report that PKA leads to AMPK activation through the LKB1 kinase. Although LKB1 has traditionally been considered a tumor suppressor, our data indicates that it may have a complex role in the thyroid gland, where its activation appears to be frequently associated with follicular thyroid carcinoma in both mice and humans.
The interplay between AMPA-type glutamate receptors (AMPARs) and major histocompatibility complex class I (MHC-I) proteins in regulating synaptic signaling is a crucial aspect of central nervous ...system (CNS) function. In this study, we investigate the significance of the cytoplasmic tail of MHC-I in synaptic signaling within the CNS and its impact on the modulation of synaptic glutamate receptor expression. Specifically, we focus on the Y321 to F substitution (Y321F) within the conserved cytoplasmic tyrosine YXXΦ motif, known for its dual role in endocytosis and cellular signaling of MHC-I. Our findings reveal that the Y321F substitution influences the expression of AMPAR subunits GluA2/3 and leads to alterations in the phosphorylation of key kinases, including Fyn, Lyn, p38, ERK1/2, JNK1/2/3, and p70 S6 kinase. These data illuminate the crucial role of MHC-I in AMPAR function and present a novel mechanism by which MHC-I integrates extracellular cues to modulate synaptic plasticity in neurons, which ultimately underpins learning and memory.
Cell-based cancer immunotherapy has achieved significant advancements, providing a source of hope for cancer patients. Notwithstanding the considerable progress in cell-based immunotherapy, the ...persistently low response rates and the exorbitant costs associated with their implementation still present a formidable challenge in clinical settings. In the landscape of cell-based cancer immunotherapies, an uncharted territory involves Type 2 innate lymphoid cells (ILC2s) and interleukin-33 (IL-33) which promotes ILC2 functionality, recognized for their inherent ability to enhance immune responses. Recent discoveries regarding their role in actuating cytolytic T lymphocyte responses, including curbing tumor growth rates and hindering metastasis, have added a new dimension to our understanding of the IL-33/ILC2 axis. These recent insights may hold significant promise for ILC2 cell-based immunotherapy. Nevertheless, the prospect of adoptively transferring ILC2s to confer immune protection against tumors has yet to be investigated. The present study addresses this hypothesis, revealing that ILC2s isolated from the lungs of tumor-bearing mice, and tumor infiltrating ILC2s when adoptively transferred after tumor establishment at a ratio of one ILC2 per sixty tumor cells, leads to an influx of tumor infiltrating CD4+ and CD8+ T lymphocytes as well as tumor infiltrating eosinophils resulting in a remarkable reduction in tumor growth. Moreover, we find that post-adoptive transfer of ILC2s, the number of tumor infiltrating ILC2s is inversely proportional to tumor size. Finally, we find corollaries of the IL-33/ILC2 axis enhancing the infiltration of eosinophils in human prostate carcinomas patients' expressing high levels of IL-33 versus those expressing low levels of IL-33. Our results underscore the heightened efficacy of adoptively transferred ILC2s compared to alternative approaches, revealing an approximately one hundred fifty-fold superiority on a cell-per-cell basis over CAR T-cells in the specific targeting and elimination of tumors within the same experimental model. Overall, this study demonstrates the functional significance of ILC2s in cancer immunosurveillance and provides the proof of concept of the potential utility of ILC2 cell-based cancer immunotherapies.
BackgroundProgrammed cell death ligand-1 (PD-L1) overexpression in tumor cells inhibits T cells activity and delivers pathologic intracellular signals that can reduce cancer treatment responses in ...pre-clinicalmodels.1 2 MethodsTo reduce tumor intracellular PD-L1-mediated pathology, we performed a drug screen that identified chlorambucil as a tumor cell PD-L1 depletion drug.ResultsChlorambucil depletes basal tumor PD-L1 expression through the ubiquitination proteasome pathway. In the tumor microenvironment, high chlorambucil doses treated orthotopic B16 melanoma and ID8agg ovarian cancer. Chlorambucil treatment efficacy was lost or reduced in PD-L1lo ID8agg and PD-L1KO B16 tumors, corroborated with in vitro data. These data suggest that chlorambucil anti-tumor activity of CAMB requires tumor PD-L1 expression, confirmed in PD-L1KO host challenge with CTRL tumor, which chlorambucil treated effectively. Chlorambucil rendered αPD-L1 resistant CTRL ID8agg and PD-L1lo B16 tumors αPD-L1 sensitive, preliminarily possibly due to tumor STING activation, and associated with enhanced tumor NK cell infiltration and central memory T cell generation. Chlorambucil also phenocopied genetic PD-L1KO by reducing tumor cell mTORC1 signals and stem cell content,3 suggesting additional treatment potential.ConclusionsChlorambucil could be a useful strategy to reprogram tumor PD-L1 signals and boost immune-based therapies especially for anti-PD-L1-resistant tumors.ReferencesClark CA et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res 2016;76:6964–6974.Juneja VR et al. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med 2017;214:895–904.Gupta HB et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1:2095–9907.Ethics ApprovalWe received approval from the UT Health San Antonio Institutional Animal Care and Use Committee (IACUC) for each procedure that used mice. We conducted each experiment per the standards required by the UT Health San Antonio Department of Laboratory Animal Resources.
BackgroundAlthough immunotherapy can induce durable anti-tumor response in multiple cancers, immune checkpoint blockade (ICB) therapy resistance in ovarian cancer and melanoma remains problematic. ...Here, we report that tumor cell-intrinsic mTORC1 regulates ICB response through mTORC1 defining subunit Raptor (Rptor) by modulating interferon-gamma (IFNg) resistance and tumor-initiating cell (TIC) virulence.MethodsWe knocked down two distinct mTORC1 signaling components: Rptor (Rptorlo, aids in mTORC1 assembly) and Lamtor1 (Ltor1lo, docks mTORC1 on lysosomes) in murine ovarian cancer ID8agg and melanoma B16 cells. PD-L1 was CRISPR knocked out in B16 and human ovarian cancer line ES2. Mice with tumors were treated with a-PD-L1± a-CD8 antibody. TICs were estimated by flow-cytometry.1 ResultsRptorlo B16 and ID8agg, but not Ltor1lo B16 tumors grew slower and were a-PD-L1 responsive unlike control (ctrl) in WT mice. We noted that ctrl and Rptorlo B16 and ID8agg cells expressed similar surface PD-L1 in vitro. Thus, Rptor suppresses a-PD-L1 response in ICB-resistant tumors. Tumor immune analysis revealed increased CD8+ T cell% and a trend to increased IFNg+CD8+ T cells in a-PD-L1 treated Rptorlo, but not ctrl B16. Rptorlo a-PD-L1 efficacy was lost with a-CD8 and in IFNg knockout mice. In vitro, IFNg suppressed Rptorlo ID8agg proliferation, unlike ctrl. These data suggested that lack of Rptor makes tumors ICB responsive, possibly by making tumors IFNg-sensitive and increasing IFNg+CD8+ T cells. Further, tumor and draining lymph node (DLN) TCF1+PD-1+ T cell stem cells (critical for aPD-L1/PD-1 success2 3) were significantly higher in a-PD-L1 treated Rptorlo tumors. Thus, tumor Rptor status could regulate tumor microenvironment and distal DLN immune landscape on a-PD-L1 treatment.We previously published that mTORC1 promotes PD-L1-dependent tumor proliferation, TIC virulence1 4 PD-L1KO B16 and ES2 cells expressed similar total Rptor protein. However, lower levels of Rptor were loaded in mTOR complex in absence of PD-L1, as assessed by a-mTOR immunoprecipitation, suggesting that pro-tumorigenic Rptor functions were downstream of, and dependent on PD-L1. Successful Rptorlo aPD-L1 treatment reduced TIC in vivo, an effect reversed in absence of CD8+ T cells or host IFNg. Inhibiting ID8agg mTORC1 with rapamycin reduced stemness genes oct4, nanog expression by QPCR. Further, ID8agg Rptorlo TIC formed significantly smaller tumors versus ctrl TIC in immune-compromised NSG mice, confirming their reduced virulence. Rptor, but not Ltor1, expression inversely correlated with tumor CD8+ infiltrate in IMvigor210 trial, and strongly with TIC gene signature in ovarian cancer patients.5 6 ConclusionsTumor-cell intrinsic Rptor modulates ICB resistance, IFNg responsiveness, immune microenvironment, and TIC virulence.AcknowledgementsN/ATrial RegistrationN/AReferencesGupta HB, et al. Tumor cell-intrinsic PD-L1 promotes tumor-initiating cell generation and functions in melanoma and ovarian cancer. Signal Transduct Target Ther 2016;1,Article number:16030.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Dammeijer F, et al. The PD-1/PD-L1-checkpoint restrains T cell immunity in tumor-draining lymph nodes. Cancer Cell 2020;38:685–700.Clark CA, et al. Tumor-intrinsic PD-L1 signals regulate cell growth, pathogenesis, and autophagy in ovarian cancer and melanoma. Cancer Res 2016;76:6964–6974.Smith BA, et al. A human adult stem cell signature marks aggressive variants across epithelial cancers. Cell Rep 2018;24:3353–3366.Hoffman-Censits JH, et al. IMvigor 210, a phase II trial of atezolizumab (MPDL3280A) in platinum-treated locally advanced or metastatic urothelial carcinoma (mUC). J Clin Oncol 2016;34, no.2_suppl:355–355.
BackgroundTumor intracellular programmed cell death ligand-1 (PDL1) mediates pathologic signals that regulate clinical treatment responses distinctly from surface-expressed PDL1 targeted by αPDL1 ...immune checkpoint blockade antibodies.MethodsWe performed a drug screen for tumor cell PDL1 depleting drugs that identified Food and Drug Administration (FDA)-approved chlorambucil and also 9-2-(phosphonomethoxy)ethyl guanine. We used in vitro and in vivo assays to evaluate treatment and signaling effects of pharmacological tumor PDL1 depletion focused on chlorambucil as FDA approved, alone or plus αPDL1.ResultsPDL1-expressing mouse and human ovarian cancer lines and mouse melanoma were more sensitive to chlorambucil-mediated proliferation inhibition in vitro versus corresponding genetically PDL1-depleted lines. Orthotopic peritoneal PDL1-expressing ID8agg ovarian cancer and subcutaneous B16 melanoma tumors were more chlorambucil-sensitive in vivo versus corresponding genetically PDL1-depleted tumors. Chlorambucil enhanced αPDL1 efficacy in tumors otherwise αPDL1-refractory, and improved antitumor immunity and treatment efficacy in a natural killer cell-dependent manner alone and plus αPDL1. Chlorambucil-mediated PDL1 depletion was relatively tumor-cell selective in vivo, and treatment efficacy was preserved in PDL1KO hosts, demonstrating tumor PDL1-specific treatment effects. Chlorambucil induced PDL1-dependent immunogenic tumor cell death which could help explain immune contributions. Chlorambucil-mediated PDL1 reduction mechanisms were tumor cell-type-specific and involved transcriptional or post-translational mechanisms, including promoting PDL1 ubiquitination through the GSK3β/β-TRCP pathway. Chlorambucil-mediated tumor cell PDL1 depletion also phenocopied genetic PDL1 depletion in reducing tumor cell mTORC1 activation and tumor initiating cell content, and in augmenting autophagy, suggesting additional treatment potential.ConclusionsPharmacological tumor PDL1 depletion with chlorambucil targets tumor-intrinsic PDL1 signaling that mediates treatment resistance, especially in αPDL1-resistant tumors, generates PDL1-dependent tumor immunogenicity and inhibits tumor growth in immune-dependent and independent manners. It could improve treatment efficacy of selected agents in otherwise treatment-refractory, including αPDL1-refractory cancers, and is rapidly clinically translatable.
Emerging cancers are sculpted by neo-Darwinian selection for superior growth and survival but minimal immunogenicity; consequently, metastatic cancers often evolve common genetic and epigenetic ...signatures to elude immune surveillance. Immune subversion by metastatic tumours can be achieved through several mechanisms; one of the most frequently observed involves the loss of expression or mutation of genes composing the MHC-I antigen presentation machinery (APM) that yields tumours invisible to Cytotoxic T lymphocytes, the key component of the adaptive cellular immune response. Fascinating ethnographic and experimental findings indicate that cannabinoids inhibit the growth and progression of several categories of cancer; however, the mechanisms underlying these observations remain clouded in uncertainty. Here, we screened a library of cannabinoid compounds and found molecular selectivity amongst specific cannabinoids, where related molecules such as Δ9-tetrahydrocannabinol, cannabidiol, and cannabigerol can reverse the metastatic immune escape phenotype
by inducing MHC-I cell surface expression in a wide variety of metastatic tumours that subsequently sensitizing tumours to T lymphocyte recognition. Remarkably, H3K27Ac ChIPseq analysis established that cannabigerol and gamma interferon induce overlapping epigenetic signatures and key gene pathways in metastatic tumours related to cellular senescence, as well as APM genes involved in revealing metastatic tumours to the adaptive immune response. Overall, the data suggest that specific cannabinoids may have utility in cancer immunotherapy regimens by overcoming immune escape and augmenting cancer immune surveillance in metastatic disease. Finally, the fundamental discovery of the ability of cannabinoids to alter epigenetic programs may help elucidate many of the pleiotropic medicinal effects of cannabinoids on human physiology.
Abstract
Tumor-intrinsic PD-L1 signals contribute to tumor virulence and pathology, but mechanisms are poorly understood. We previously showed that tumor-intrinsic PD-L1 increases mTORC1 and tumor ...initiating cell (TIC) generation and function in ovarian cancer and melanoma. We used CRISPR/Cas9 (KO) and shRNA (lo) to reduce PD-L1 in mouse melanoma (B16) and ovarian cancer (ID8agg), and human ovarian cancer (ES2). PD-L1lo reduced mTORC1 and TIC as expected, but unexpectedly increased actin polymerization and filopodia, and altered cell morphology. The actin depolymerizing agents Latrunculin-A or Cytochalasin-D reversed actin polymerization in PD-L1lo ID8agg cells and increased mTORC1 (p-rpS6) without affecting mTORC2 (pAktS473). Latrunculin-A and Cytochalasin-D increased TIC numbers and stemness gene expression (Oct4) in PD-L1lo ID8agg cells with no effect on control ID8agg cells. Similar actin effects were seen in PD-L1KO B16 and PD-L1KO ES2. To confirm TIC function, Latrunculin-A increased tumorospheres (TIC self-renewal function) in PD-L1lo, but not control ID8agg cells. The mTORC1 inhibitor rapamycin reduced TIC numbers and functions with no effect on actin polymerization, suggesting mTORC1 is downstream of actin polymerization. Rptorlo ID8agg cells (with low mTORC1 signaling) did not exhibit the PD-L1lo actin polymerization, nor did Latrunculin-A increase Rptorlo ID8agg TIC numbers, tumorospheres, or stemness genes, confirming the flow of signals is PD-L1 to actin to mTORC1 to TIC (stemness). Our data define a highly novel tumor virulence control mechanism of cell-intrinsic PD-L1 through inhibiting actin polymerization suggesting new drug discovery targets. Preliminary data show some similar effects of tumor PD-1.
Abstract
Rearranged in transformation/ papillary thyroid carcinoma 3 (RET/PTC3 or RP3), is a unique fusion oncogene, responsible for a benign version of PTC. RP3 has an active inflammatory component ...leading to the secretion of various pro-inflammatory cytokines, however the role of inflammation in development and progression of PTC has not been clearly defined. Facilitating investigation of this issue is our finding that the transforming and inflammatory signals of RP3 can be functionally separated allowing us to study each in isolation. We generated several RP3 mutants ablated in; 1) transformative signaling due to mutation of a tyrosine residue (RP3Y588Fmutant) which prevents the binding of downstream adaptor proteins 2) inflammatory signaling through mutation of binding sites for TRAF2 and TRAF6. Transformation only RP3 mutants grew tumors in immunodeficient and immunocompetent mice, strengthening our hypothesis that the inflammatory component of RP3 is responsible for the benign nature of PTC.
To assess the impact of inflammatory RP3 on the progression of different tumors, various thyroid and non-thyroid tumorigenic cell lines were transduced with MSCV_IRES_GFP_RP3 mutants and sorted for GFP expression. With highly immunogenic MC57G cells (C57BL/6 mice), the transforming only mutants developed tumors whereas wild type RP3 along with inflammatory only RP3 were rejected. Inflammation only RP3 mutant also led to slower tumor growth in Renca cell line (Balb/c) demonstrating the effect to be independent of haplotype. We are currently extending the analysis to PTC lines with BRAF and KRAS mutations, which are usually associated with more aggressive tumor profiles without an inflammatory tumor microenvironment. Our data so far suggest that contrary to the contemporary view, inflammation can retard tumor progression. Thus, the RP3 oncogene might be exploited in this regard. Future experiments aim to further characterize the importance of RP3-driven inflammation in tumor growth through continued in vivo and in vitro studies.
Citation Format: Suresh C. Kari, Laurence C. Eisenlohr. Impact of RET/PTC3 oncogene's inflammatory and transformative signaling on tumorigenicity of thyroid and non-thyroid tumorigenic cell lines. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4077. doi:10.1158/1538-7445.AM2014-4077
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