Erratum: The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

[This corrects the article on p. 2621 in vol. 10, PMID: 32905506.].

The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

The immune checkpoint blockade therapy has emerged as encouraging treatment strategies in various cancer types. Anti-PD-L1 (programmed death-ligand 1) antibodies have been approved for triple-negative breast cancer, however the response rate yet to be optimized. It would be imperative to further understand and investigate the molecular mechanisms of PD-L1 regulation. Here, we identified glucose regulatory protein 78 (GRP78), a major endoplasmic reticulum (ER) stress responding protein, as a novel binding partner of PD-L1. GRP78 interacts with PD-L1 at the ER region and increases PD-L1 levels via regulating its stability. ER stress, triggered by different stimuli such as conventional chemotherapy, leads to the induction of PD-L1 in a GRP78-dependent manner. We showed that GRP78 modulates the response to chemotherapy, and dual-high levels of GRP78 and PD-L1 correlates with poor relapse-free survival in triple-negative breast cancer. Altogether, our study provides novel molecular insights into the regulatory mechanism of PD-L1 by revealing its interaction with GRP78, and offers a rationale to target GRP78 as a potential therapeutic strategy to enhance anti-tumor immunity.

Inhibition of ATR downregulates PD-L1 and sensitizes tumor cells to T cell-mediated killing.

The ataxia telangiectasia and Rad3-related (ATR) kinase plays a crucial role in maintaining genome stability in response to DNA damage. Once activated, ATR acts via its downstream target to arrest the cell cycle, promote DNA repair, and enhance cell survival. Therefore, ATR has become an attractive therapeutic target in cancer therapy. Multiple clinical studies have demonstrated that ATR inhibitors can sensitize cancer cells to conventional DNA damaging agents. However, the potential effects of ATR inhibitors on immune response in the tumor microenvironment, especially on the expression of immune checkpoint-related proteins, remain elusive. Here we show that DNA damaging agents, such as ionizing radiation and cisplatin, significantly induce cell surface PD-L1 expression in various cancer cell types. This effect is blocked by depletion or pharmacological inhibition of ATR, suggesting the essential role of ATR in DNA damage-induced PD-L1 expression. Mechanistically, we show that disruption of ATR destabilizes PD-L1 in a proteasome-dependent manner. Furthermore, clinical ATR kinase inhibitor downregulates PD-L1 expression to attenuate PD-L1/PD-1 interaction and sensitize cancer cells to T cell killing. Collectively, our findings indicate that in addition to potentiating DNA damage, ATR inhibitor concurrently downregulates PD-L1 levels and enhances anti-tumor immune responses. Moreover, our data reveal a potential crosstalk between DNA damage response signaling and immune checkpoints, providing a rationale for the combination therapy of ATR inhibitor and immune checkpoint blockade.

Galectin-12 in Cellular Differentiation, Apoptosis and Polarization.

Galectin-12 is a member of a family of mammalian lectins characterized by their affinity for ß-galactosides and consensus amino acid sequences. The protein structure consists of a single polypeptide chain containing two carbohydrate-recognition domains joined by a linker region. Galectin-12 is predominantly expressed in adipose tissue, but is also detected in macrophages and other leukocytes. Downregulation of galectin-12 in mouse 3T3-L1 cells impairs their differentiation into adipocytes. Conversely, overexpression of galectin-12 in vitro induces cell cycle arrest in G1 and apoptosis. Upregulation of galectin-12 and initiation of G1 cell cycle arrest are associated with driving pre-adipocytes toward terminal differentiation. Galectin-12 deficiency increases insulin sensitivity and glucose tolerance in obese animals. Galectin-12 inhibits macrophage polarization to the M2 population, enhancing inflammation and decreasing insulin sensitivity in adipocytes. Galectin-12 also affects myeloid differentiation, which is associated with chemotherapy resistance. In addition to highlighting the above-mentioned aspects, this review also discusses the potential clinical applications of modulating the function of galectin-12.

Galectin-12 inhibits granulocytic differentiation of human NB4 promyelocytic leukemia cells while promoting lipogenesis.

As a member of the galectin family of animal lectins, galectin-12 is preferentially expressed in adipocytes and leukocytes. In adipocytes, galectin-12 is associated with lipid droplets and regulates lipid metabolism and energy balance, whereas its role in leukocytes is not clear. Analysis of galectin-12 expression in a public data set of acute myeloid leukemia (AML) samples revealed that it is selectively overexpressed in the M3 subtype, which is also known as acute promyelocytic leukemia (APL). To investigate the role of galectin-12 in APL cells, we manipulated its expression in the APL cell line, NB4, and measured resultant effects on all-trans-retinoic acid (ATRA)-induced granulocytic differentiation. With a doxycycline-inducible gene knockdown system, we found that suppression of galectin-12 promoted ATRA-induced neutrophil differentiation but inhibited lipid droplet formation. Our results indicate that overexpression of galectin-12 contributes to a differentiation block in APL cells, and suppression of galectin-12 facilitates granulocytic differentiation. Furthermore, these data suggest that lipogenesis and other aspects of myeloid differentiation can be differentially regulated. Taken together, these findings suggest that galectin-12 may be a target for treatment of the ATRA-resistant subset of APL.

Analysis of the intracellular role of galectins in cell growth and apoptosis.

Galectins are a family of animal lectins with conserved carbohydrate-recognition domains that recognize ß-galactosides. Despite structural similarities, these proteins have diverse functions in a variety of cellular processes. While a large number of extracellular functions have been demonstrated for galectins, the existence of intracellular functions has been clearly shown for a number of galectins, including regulation of cell growth and apoptosis; these latter functions may not involve glycan binding. There is considerable interest in intracellular regulation by galectins of cell growth and apoptosis, as these are fundamental cellular processes in normal homeostasis. Their dysregulation can cause pathologies such as autoimmune disorders, cancer, and neural degenerative diseases. Here we describe methods that we routinely perform in the laboratory to investigate the role of galectins in cell growth and apoptosis. These include methods for cell isolation, cell maintenance, and genetic manipulations to perturb galectin gene expression, as well as assays for cell growth and apoptosis.

Galectin-3 regulates intracellular trafficking of EGFR through Alix and promotes keratinocyte migration.

The EGFR-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that the absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are markedly reduced, and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this previously unreported function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the ESCRT (endosomal sorting complex required for transport) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors.

Ablation of a galectin preferentially expressed in adipocytes increases lipolysis, reduces adiposity, and improves insulin sensitivity in mice.

The breakdown of triglycerides, or lipolysis, is a tightly controlled process that regulates fat mobilization in accord with an animal's energy needs. It is well established that lipolysis is stimulated by hormones that signal energy demand and is suppressed by the antilipolytic hormone insulin. However, much still remains to be learned about regulation of lipolysis by intracellular signaling pathways in adipocytes. Here we show that galectin-12, a member of a ß-galactoside-binding lectin family preferentially expressed by adipocytes, functions as an intrinsic negative regulator of lipolysis. Galectin-12 is primarily localized on lipid droplets and regulates lipolytic protein kinase A signaling by acting upstream of phosphodiesterase activity to control cAMP levels. Ablation of galectin-12 in mice results in increased adipocyte mitochondrial respiration, reduced adiposity, and ameliorated insulin resistance/glucose intolerance. This study identifies unique properties of this intracellular galectin that is localized to an organelle and performs a critical function in lipid metabolism. These findings add to the significant functions exhibited by intracellular galectins, and have important therapeutic implications for human metabolic disorders.

Galectins: structure, function and therapeutic potential.

Galectins are a family of animal lectins that bind beta-galactosides. Outside the cell, galectins bind to cell-surface and extracellular matrix glycans and thereby affect a variety of cellular processes. However, galectins are also detectable in the cytosol and nucleus, and may influence cellular functions such as intracellular signalling pathways through protein-protein interactions with other cytoplasmic and nuclear proteins. Current research indicates that galectins play important roles in diverse physiological and pathological processes, including immune and inflammatory responses, tumour development and progression, neural degeneration, atherosclerosis, diabetes, and wound repair. Some of these have been discovered or confirmed by using genetically engineered mice deficient in a particular galectin. Thus, galectins may be a therapeutic target or employed as therapeutic agents for inflammatory diseases, cancers and several other diseases.

Galectin-7 (PIG1) exhibits pro-apoptotic function through JNK activation and mitochondrial cytochrome c release.

Galectin-7 is normally expressed in all types of stratified epithelia, but is significantly down-regulated in squamous cell carcinomas. This protein was recently found to be highly inducible by p53 in a colon carcinoma cell line, DLD-1, and designated as PIG1 (for p53-induced gene 1). We studied transfectants of HeLa and DLD-1 cells ectopically expressing this protein and found that they were more susceptible to apoptosis than control transfectants. This was observed in apoptosis induced by mechanistically distinct stimuli, suggesting that galectin-7 acts on a common point in the apoptosis signaling pathways. Further analyses of actinomycin D-induced apoptosis demonstrated that galectin-7 expression causes enhanced caspase-3 activity and poly(ADP-ribose) polymerase cleavage, and the potentiation of apoptosis by galectin-7 was completely abrogated by a caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. In addition, galectin-7 transfectants displayed accelerated mitochondrial cytochrome c release and up-regulated JNK activity upon apoptosis induction. Several lines of evidence indicate that the effect on apoptosis is not due to the lectin functioning extracellularly through interactions with cell surface glycoconjugates. In fact, this lectin is found to localize in nuclei and cytoplasm of the transfectants and the transformed keratinocyte line HaCaT. Therefore, galectin-7 is a pro-apoptotic protein that functions intracellularly upstream of JNK activation and cytochrome c release. DNA microarray analysis revealed genes that are differentially expressed between galectin-7 and control transfectants. Some of them are potentially contributory to this lectin's proapoptotic function and these include redox-related genes monoamine oxidase B, ryanodine receptor 2, and glutathione S-transferase Mu 3.