Rab37 mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion in lung cancer.

BACKGROUND: Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of T cells. High expression of PD-1 leads to T-cell dysfunction in the tumor microenvironment (TME). However, the mechanism of intracellular trafficking and plasma membrane presentation of PD-1 remains unclear. METHODS: Multiple databases of lung cancer patients were integratively analyzed to screen Rab proteins and potential immune-related signaling pathways. Imaging and various biochemical assays were performed in Jurkat T cells, splenocytes, and human peripheral blood mononuclear cells (PBMCs). Rab37 knockout mice and specimens of lung cancer patients were used to validate the concept. RESULTS: Here, we identify novel mechanisms of intracellular trafficking and plasma membrane presentation of PD-1 mediated by Rab37 small GTPase to sustain T cell exhaustion, thereby leading to poor patient outcome. PD-1 colocalized with Rab37-specific vesicles of T cells in a GTP-dependent manner whereby Rab37 mediated dynamic trafficking and membrane presentation of PD-1. However, glycosylation mutant PD-1 delayed cargo recruitment to the Rab37 vesicles, thus stalling membrane presentation. Notably, T cell proliferation and activity were upregulated in tumor-infiltrating T cells from the tumor-bearing Rab37 knockout mice compared to those from wild type. Clinically, the multiplex immunofluorescence-immunohistochemical assay indicated that patients with high Rab37+/PD-1+/TIM3+/CD8+ tumor infiltrating T cell profile correlated with advanced tumor stages and poor overall survival. Moreover, human PBMCs from patients demonstrated high expression of Rab37, which positively correlated with elevated levels of PD-1+ and TIM3+ in CD8+ T cells exhibiting reduced tumoricidal activity. CONCLUSIONS: Our results provide the first evidence that Rab37 small GTPase mediates trafficking and membrane presentation of PD-1 to sustain T cell exhaustion, and the tumor promoting function of Rab37/PD-1 axis in T cells of TME in lung cancer. The expression profile of Rab37high/PD-1high/TIM3high in tumor-infiltrating CD8+ T cells is a biomarker for poor prognosis in lung cancer patients.

Autophagy drives plasticity and functional polarization of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and are key cells in regulating tumor development, metastasis, immune responses, inflammation, and chemoresistance. In response to TME stimulation, circulating monocytes are recruited and differentiated as TAMs. Most TAMs are defined as alternatively activated (M2) phenotype to create immunosuppressive TME and support tumor progression. In contrast, classically activated (M1) TAMs can produce pro-inflammatory cytokines and enhance immune responses against tumor development. Autophagy is a conserved catabolic process to control cellular homeostasis and biological function. Emerging evidence reveals crucial contribution of autophagy in modulating TAM plasticity and functional polarization in TME. In this review, we introduce the current understanding of autophagy-regulated TAM function in development of cancer. We focus on how autophagy modulates antigen presentation, LC3-associated phagocytosis, cytokine secretion, inflammasome regulation, recruitment, differentiation, and polarization of TAMs and suggest strategies for potential therapeutics by targeting autophagy in TAMs. We expect this review can provide a new notion of future cancer immunotherapy.

Recent advances in conventional and unconventional vesicular secretion pathways in the tumor microenvironment.

All cells in the changing tumor microenvironment (TME) need a class of checkpoints to regulate the balance among exocytosis, endocytosis, recycling and degradation. The vesicular trafficking and secretion pathways regulated by the small Rab GTPases and their effectors convey cell growth and migration signals and function as meditators of intercellular communication and molecular transfer. Recent advances suggest that Rab proteins govern conventional and unconventional vesicular secretion pathways by trafficking widely diverse cargoes and substrates in remodeling TME. The mechanisms underlying the regulation of conventional and unconventional vesicular secretion pathways, their action modes and impacts on the cancer and stromal cells have been the focus of much attention for the past two decades. In this review, we discuss the current understanding of vesicular secretion pathways in TME. We begin with an overview of the structure, regulation, substrate recognition and subcellular localization of vesicular secretion pathways. We then systematically discuss how the three fundamental vesicular secretion processes respond to extracellular cues in TME. These processes are the conventional protein secretion via the endoplasmic reticulum-Golgi apparatus route and two types of unconventional protein secretion via extracellular vesicles and secretory autophagy. The latest advances and future directions in vesicular secretion-involved interplays between tumor cells, stromal cell and host immunity are also described.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction.

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Converged Rab37/IL-6 trafficking and STAT3/PD-1 transcription axes elicit an immunosuppressive lung tumor microenvironment.

Background: Increased IL-6 level, M2 macrophages and PD-1+CD8+ T cells in tumor microenvironments (TME) have been identified to correlate with resistance to checkpoint blockade immunotherapy, yet the mechanism remains poorly understood. Rab small GTPase-mediated trafficking of cytokines is critical in immuno-modulation. We have previously reported dysregulation of Rab37 in lung cancer cells, whereas the roles of Rab37 in tumor-infiltrating immune cells and cancer immunotherapy are unclear. Methods: The tumor growth of the syngeneic mouse allograft in wild type or Rab37 knockout mice was analyzed. Imaging analyses and vesicle isolation were conducted to determine Rab37-mediated IL-6 secretion. STAT3 binding sites at PD-1 promoter in T cells were identified by chromatin immunoprecipitation assay. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37, IL-6 and PD-1 and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from lung cancer patients. Results: We revealed that Rab37 regulates the secretion of IL-6 in a GTPase-dependent manner in macrophages to trigger M2 polarization. Macrophage-derived IL-6 promotes STAT3-dependent PD-1 mRNA expression in CD8+ T cells. Clinically, tumors with high stromal Rab37 and IL-6 expression coincide with tumor infiltrating M2-macrophages and PD1+CD8+ T cells that predicts poor prognosis in lung cancer patients. In addition, lung cancer patients with an increase in plasma IL-6 level are found to be associated with immunotherapeutic resistance. Importantly, combined blockade of IL-6 and CTLA-4 improves survival of tumor-bearing mice by reducing infiltration of PD1+CD8+ T cells and M2 macrophages in TME. Conclusions: Rab37/IL-6 trafficking pathway links with IL-6/STAT3/PD-1 transcription regulation to foster an immunosuppressive TME and combined IL-6/CTLA-4 blockade therapy exerts potent anti-tumor efficacy.

Autophagy Drives Galectin-1 Secretion From Tumor-Associated Macrophages Facilitating Hepatocellular Carcinoma Progression.

Galectin-1 (Gal-1) is a secretory lectin with pro-tumor activities and is associated strongly with hepatocellular carcinoma (HCC) development. Although Gal-1 is a well-known soluble pro-tumor factor in the tumor microenvironment (TME), the secretion mode of Gal-1 is not clearly defined. On the other hand, in addition to cancer cells, Gal-1 is widely expressed in tumor stromal cells, including tumor-associated macrophages (TAMs). TAMs are a significant component of stromal cells in TME; however, their contributions in producing Gal-1 to TME are still not explored. Here we reveal that TAMs can actively secrete Gal-1 in response to stimuli of HCC cells. Gal-1 produced by TAMs leads to an increase of the systemic level of Gal-1 and HCC tumor growth in mice. Mechanistically, TLR2-dependent secretory autophagy is found to be responsible for Gal-1 secretion from TAMs. Gal-1 acts as a cargo of autophagosomes to fuse with multivesicular bodies via Rab11 and VAMP7-mediated vesicle trafficking before being secreted. This autophagy-regulated Gal-1 secretion in TAMs correlates to poor overall survival and progression-free survival rates of HCC patients. Our findings uncover the secretion mode of Gal-1 via secretory autophagy and highlight the pathological role of TAM-produced Gal-1 in HCC progression.

Hepatocellular carcinoma-derived high mobility group box 1 triggers M2 macrophage polarization via a TLR2/NOX2/autophagy axis.

In many human cancers, including hepatocellular carcinoma (HCC), high density of infiltrating tumor-associated macrophages (TAM) is associated with poor prognosis. Most TAMs express a M2 phenotype subsequently supporting tumor growth. How tumor cells polarize these TAMs to a pro-tumor M2 phenotype is still poorly understood. Our previous studies have revealed that a Toll-like receptor 2 (TLR2)-dependent autophagy triggered by hepatoma-derived factors down-regulates NF-κB p65 and drives M2 macrophage differentiation. However, the underlying mechanisms and potential hepatoma-derived TLR2 ligands are not clear. Here, we provide evidence to reveal that NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS) generation is crucial for HCC-induced autophagy, NF-κB p65 down-regulation and M2 phenotype polarization in primary macrophages. This NOX2-generated ROS production in abolished in TLR2-deficient macrophages. HCC-derived or recombinant high-mobility group box 1 (HMGB1) is able to trigger this TLR2-mediated M2 macrophage polarization. Blockage of HMGB1 and ROS by inhibitors, ethyl pyruvate and N-acetylcysteine amide, respectively, significantly reduces both M2 macrophage accumulation and liver nodule formation in HCC-bearing mice. Our findings uncover a HMGB1/TLR2/NOX2/autophagy axis to trigger M2 macrophage polarization in HCC that can be considered as a novel therapeutic target for treating HCC.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Evaluation of Anti-aging Compounds Using the Promoters of Elastin and Fibrillin-1 Genes Combined with a Secreted Alkaline Phosphatase Reporter in Normal Human Fibroblasts.

Elastic fibers are major constituents of the extracellular matrix (ECM) in dynamic tissues in the human body, and regulation of elastin and fibrillin-1 expression mediates the formation of these fibers. Traditional assays for the measurement of elastin and fibrillin-1, such as western blotting, Luna staining and immunostaining, are relatively complex and time-consuming. Thus, a relatively simple assay system that also provides rational results is urgently needed. In the study, we aimed to develop a human cell-based assay system that can be used to analyze functional compounds using the promoters of elastin (ELN) and fibrillin-1 (FBN1) genes integrated with a secreted alkaline phosphatase (SEAP) reporter in normal human fibroblast cells. We used this system to assess anti-aging compounds. We used several regulators of elastinogenesis, including retinol, coenzyme Q10, deoxyArbutin and Elestan(TM) (Manilkara multinervis leaf extract), to verify the efficacy of this assay system. Our results demonstrate that this assay system can be used as a fast and realistic method for identifying anti-aging components for future use in foods, cosmetics and drugs.

Cyclin B1 destruction box-mediated protein instability: the enhanced sensitivity of fluorescent-protein-based reporter gene system.

The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.