Cancer-associated mutations in protein kinase C theta are loss-of-function.

The Ca2+-independent, but diacylglycerol-regulated, novel protein kinase C (PKC) theta (θ) is highly expressed in hematopoietic cells where it participates in immune signaling and platelet function. Mounting evidence suggests that PKCθ may be involved in cancer, particularly blood cancers, breast cancer, and gastrointestinal stromal tumors, yet how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Here, we examine the effect of four cancer-associated mutations, R145H/C in the autoinhibitory pseudosubstrate, E161K in the regulatory C1A domain, and R635W in the regulatory C-terminal tail, on the cellular activity and stability of PKCθ. Live-cell imaging studies using the genetically-encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter 2 (CKAR2), revealed that the pseudosubstrate and C1A domain mutations impaired autoinhibition to increase basal signaling. This impaired autoinhibition resulted in decreased stability of the protein, consistent with the well-characterized behavior of Ca2+-regulated PKC isozymes wherein mutations that impair autoinhibition are paradoxically loss-of-function because the mutant protein is degraded. In marked contrast, the C-terminal tail mutation resulted in enhanced autoinhibition and enhanced stability. Thus, the examined mutations were loss-of-function by different mechanisms: mutations that impaired autoinhibition promoted the degradation of PKC, and those that enhanced autoinhibition stabilized an inactive PKC. Supporting a general loss-of-function of PKCθ in cancer, bioinformatics analysis revealed that protein levels of PKCθ are reduced in diverse cancers, including lung, renal, head and neck, and pancreatic. Our results reveal that PKCθ function is lost in cancer.

PKC-θ is an important driver of fluoride-induced immune imbalance of regulatory T cells/effector T cells.

Fluoride is unnecessary in the human body. Long-term fluoride exposure may lead to immune system abnormalities. However, the mechanism remains unclear. This study aim to explore the mechanism of fluoride interference in the immune system and also identify the key indicators of fluoride-induced immune damage. Questionnaires were used to collect basic information. Multiple linear analyses and other statistical methods were used in order to process the data. Flow cytometry was used to detect relevant immunomarkers and analyze immune damage. Simultaneously, Wistar rats and cell models exposed to fluoride were established to detect the effects of fluoride on immune homeostasis. The results showed that sex, residence time, smoking, and Corona Virus Disease 2019 (COVID-19) infection may indirectly influence fluoride-induced immune damage. In residents of fluoride-exposed areas, there was a significant decrease in CD3+ T lymphocytes and CD4+ and CD8+ cells and a downward trend in the CD4+/CD8+ cell ratio. CD4+CD8+/CD4+, regulatory T cells (Tregs), and Tregs/effector T cells (Teffs) ratios showed opposite changes. Fluoride inhibits T cell activation by inhibiting the expression and phosphorylation of Protein Kinase C-θ (PKC-θ), hinders the internalization of T cell receptors, and affects NF-kB and c-Jun protein expression, leading to homeostatic Treg/Teff imbalance in vivo and in vitro experiments. This study represents the first evidence suggesting that PKC-θ may be the key to immune imbalance in the body under fluoride exposure. It is possible that Tregs/Teffs cell ratio provide a reference point for the diagnosis and treatment of fluoride-induced immune damage.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

ABSTRACT: The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

Fasting regulates mitochondrial function through lncRNA PRKCQ-AS1-mediated IGF2BPs in papillary thyroid carcinoma.

Recurring evidence suggests that fasting has extensive antitumor effects in various cancers, including papillary thyroid carcinoma (PTC). However, the underlying mechanism of this relationship with PTC is unknown. In this study, we study the effect of fasting on glycolysis and mitochondrial function in PTC. We find that fasting impairs glycolysis and reduces mitochondrial dysfunction in vitro and in vivo and also fasting in vitro and fasting mimicking diets (FMD) in vivo significantly increase the expression of lncRNA-protein kinase C theta antisense RNA 1 (PRKCQ-AS1), during the inhibition of TPC cell glycolysis and mitochondrial function. Moreover, lncRNA PRKCQ-AS1 was significantly lower in PTC tissues and cells. In addition, PRKCQ-AS1 overexpression increased PTC cell glycolysis and mitochondrial function; PRKCQ-AS1 knockdown has the opposite effect. On further mechanistic analysis, we identified that PRKCQ-AS1 physically interacts with IGF2BPs and enhances protein arginine methyltransferases 7 (PRMT7) mRNA, which is the key player in regulating glycolysis and mitochondrial function in PTC. Hence, PRKCQ-AS1 inhibits tumor growth while regulating glycolysis and mitochondrial functions via IGF2BPs/PRMT7 signaling. These results indicate that lncRNA PRKCQ-AS1 is a key downstream target of fasting and is involved in PTC metabolic reprogramming. Further, the PRKCQ-AS1/IGF2BPs/PRMT7 axis is an ideal therapeutic target for PTC diagnosis and treatment.

N-degron-mediated degradation of the proteolytically activated form of PKC-theta kinase attenuates its pro-apoptotic function.

Cellular signalling cues lead to the initiation of apoptotic pathways and often result in the activation of caspases which in turn cause the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N-degron degradation pathways. Here, we investigate the proteolytically generated fragment of the PKC theta kinase, where we demonstrate the first report on the stability of this pro-apoptotic protein fragment. We have determined that the pro-apoptotic cleaved fragment of PKC-theta is unstable in cells because its N-terminal lysine targets it for proteasomal degradation via the N-degron degradation pathway and this degradation is inhibited by mutating the destabilizing N-termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC-theta or inhibition of the N-degron degradation augments the apoptosis-inducing effect of staurosporine in Jurkat cells. Notably, we have unveiled that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T-cell leukemia. Our results expand the functional scope of mammalian N-degron degradation pathways, and support the notion that targeting N-degron degradation machinery may have promising therapeutic implications in cancer cells.

MiR-128-1-5p inhibits cell proliferation and induces cell apoptosis via targeting PRKCQ in colorectal cancer.

Previous studies have indicated that miR-128 was downregulated in a variety of cancers including colorectal cancer (CRC). However, the role and the underlying molecular mechanisms of miR-128 in CRC still remain largely unknown. The aim of this study was to investigate the level of miR-128-1-5p in CRC patients and to explore both the effects and regulatory mechanisms of miR-128-1-5p in the malignancy of CRC. Real-time PCR and western blot were used to analyze the expression levels of miR-128-1-5p and the direct downstream target protein tyrosine kinase C theta isoform (PRKCQ). Cell Counting Kit-8, clone formation, TUNEL apoptosis assays, and subcutaneous tumor model were performed to investigate the malignant ability of colon cancer cells. A luciferase assay was performed to explore whether miR-128-1-5p could directly bind to 3'-UTR region of PRKCQ. In the present study, we detected the decreased expression and clinical significances of miR-128-1-5p in colorectal cancer tissues and cell lines. Functional experiments revealed that miR-128-1-5p inhibited cell proliferation and induced cell apoptosis and that PRKCQ was identified as a target of miR-128-1-5p and involved in miR-128-1-5p-mediated proliferation and apoptosis. In conclusion, our results showed that miR-128-1-5p reduced CRC growth by modulating PRKCQ expression and is a possible new therapeutic target for patients with CRC.

LKB1 isoform expression modulates T cell plasticity downstream of PKCθ and IL-6.

Following activation, CD4 T cells undergo metabolic and transcriptional changes as they respond to external cues and differentiate into T helper (Th) cells. T cells exhibit plasticity between Th phenotypes in highly inflammatory environments, such as colitis, in which high levels of IL-6 promote plasticity between regulatory T (Treg) cells and Th17 cells. Protein Kinase C theta (PKCθ) is a T cell-specific serine/threonine kinase that promotes Th17 differentiation while negatively regulating Treg differentiation. Liver kinase B1 (LKB1), also a serine/threonine kinase and encoded by Stk11, is necessary for Treg survival and function. Stk11 can be alternatively spliced to produce a short variant (Stk11S) by transcribing a cryptic exon. However, the contribution of Stk11 splice variants to Th cell differentiation has not been previously explored. Here we show that in Th17 cells, the heterogeneous ribonucleoprotein, hnRNPLL, mediates Stk11 splicing into its short splice variant, and that Stk11S expression is diminished when Hnrnpll is depleted using siRNA knock-down approaches. We further show that PKCθ regulates hnRNPLL and, thus, Stk11S expression in Th17 cells. We provide additional evidence that exposing induced (i)Tregs to IL-6 culminates in Stk11 splicing downstream of PKCθAltogether our data reveal a yet undescribed outside-in signaling pathway initiated by IL-6, that acts through PKCθ and hnRNPLL to regulate Stk11 splice variants and facilitate Th17 cell differentiation. Furthermore, we show for the first time, that this pathway can also be initiated in developing iTregs exposed to IL-6, providing mechanistic insight into iTreg phenotypic stability and iTreg to Th17 cell plasticity.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

LncRNA PRKCQ-AS1 regulates paclitaxel resistance in triple-negative breast cancer cells through miR-361-5p/PIK3C3 mediated autophagy.

Paclitaxel (PTX) resistance is a key cause of chemotherapy failure in patients with triple negative breast cancer (TNBC). The aim of this study is to investigate the effect and mechanism of long non-coding RNA (lncRNA) on the PTX resistance of TNBC cells through autophagy. MDA-MB-231 cells are used to induce the PTX-resistant TNBC cell line MDA-MB-231.PR (MDR) by increasing dose intermittently. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the mRNA levels of phosphoinositide-3-kinase class 3 (PIK3C3), miR-361-5p and lncRNA PRKCQ-AS1 in the cells, and Western blot analysis was used to detect the protein expressions of PIK3C3, autophagy-related, drug-resistant and apoptosis-related genes. MDC staining detected the formation of autophagic vacuoles. The interactions between miR-361-5p and PIK3C3 and between lncRNA PRKCQ-AS1 and miR-361-5p were verified by dual-luciferase assay. Cell viability, apoptosis, migration and invasion were assessed by performing MTT, flow cytometry assay, and transwell assay. The mRNA level of miR-361-5p and the autophagy and drug resistance levels of TNBC PTX-resistant cells were significantly up-regulated. miR-361-5p could target autophagy-related gene PIK3C3, and overexpression of miR-361-5p could down-regulate PIK3C3 protein expression and autophagy level and PTX resistance of MDR cells. LncRNA PRKCQ-AS1 was selected through bioanalysis, and miR-361-5p could target lncRNA PRKCQ-AS1. In addition, lncRNA PRKCQ-AS1 level was up-regulated in TNBC PTX-resistant cells, and knockdown of lncRNA PRKCQ-AS1 could weaken autophagy and drug resistance level and could promote cell apoptosis. Overexpression of lncRNA PRKCQ-AS1 reversed the pro-apoptotic effect and down-regulation of autophagy and resistance levels was induced by miR-361-5p. In vivo experiments were performed to verify the role of lncRNA PRKCQ-AS1. We demonstrate that down-regulation of lncRNA PRKCQ-AS1 weakened PTX resistance and promoted cell apoptosis by miR-361-5p/PIK3C3 mediated autophagy.

Hyperprogression of cutaneous T cell lymphoma after anti-PD-1 treatment.

BACKGROUNDImmune checkpoint blockade is an emerging treatment for T cell non-Hodgkin's lymphoma (T-NHL), but some patients with T-NHL have experienced hyperprogression with undetermined mechanisms upon anti-PD-1 therapy.METHODSSingle-cell RNA-Seq, whole-genome sequencing, whole-exome sequencing, and functional assays were performed on primary malignant T cells from a patient with advanced cutaneous T cell lymphoma who experienced hyperprogression upon anti-PD-1 treatment.RESULTSThe patient was enrolled in a clinical trial of anti-PD-1 therapy and experienced disease hyperprogression. Single-cell RNA-Seq revealed that PD-1 blockade elicited a remarkable activation and proliferation of the CD4+ malignant T cells, which showed functional PD-1 expression and an exhausted status. Further analyses identified somatic amplification of PRKCQ in the malignant T cells. PRKCQ encodes PKCθ; PKCθ is a key player in the T cell activation/NF-κB pathway. PRKCQ amplification led to high expressions of PKCθ and p-PKCθ (T538) on the malignant T cells, resulting in an oncogenic activation of the T cell receptor (TCR) signaling pathway. PD-1 blockade in this patient released this signaling, derepressed the proliferation of malignant T cells, and resulted in disease hyperprogression.CONCLUSIONOur study provides real-world clinical evidence that PD-1 acts as a tumor suppressor for malignant T cells with oncogenic TCR activation.TRIAL REGISTRATIONClinicalTrials.gov (NCT03809767).FUNDINGThe National Natural Science Foundation of China (81922058), the National Science Fund for Distinguished Young Scholars (T2125002), the National Science and Technology Major Project (2019YFC1315702), the National Youth Top-Notch Talent Support Program (283812), and the Peking University Clinical Medicine plus X Youth Project (PKU2019LCXQ012) supported this work.

Development and validation of the prognostic model based on autophagy-associated genes in idiopathic pulmonary fibrosis.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease. Many studies suggest that autophagy may be related to disease progression and prognosis in IPF. However, the mechanisms involved have not been fully elucidated. Methods: We incorporated 232 autophagy-associated genes (AAGs) and two datasets, GSE28042 and GSE27957, from the GEO database. Univariate Cox analysis and least absolute shrinkage and selection operator (LASSO) regression were used to construct the autophagy-associated prognostic model. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the functions of these autophagy-associated genes. CIBERSORT algorithm was used to calculate the immune cell infiltration between patients in the high-risk score and low-risk score groups. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) was performed to explore the mRNA expression of five genes in the autophagy-associated risk model. Results: We constructed a 5-autophagy-associated genes signature based on Univariate Cox analysis and LASSO regression. In our autophagy-associated risk model, IPF patients in the high-risk group demonstrated a poor overall survival rate compared to patients in the low-risk group. For 1-, 2-, and 3-year survival rates, the AUC predictive value of the AAG signature was 0.670, 0.787, and 0.864, respectively. These results were validated in the GSE27957 cohort, confirming the good prognostic effect of our model. GO and KEGG pathway analyses enriched immune-related pathways between the high-risk and low-risk groups. And there was also a significant difference in immune cell infiltration between two groups. And the results of qRT-PCR showed that the expression levels of FOXO1, IRGM, MYC, and PRKCQ were significantly decreased in the Peripheral Blood Mononuclear Cell (PBMC) of IPF patient samples. Conclusion: Our study constructed and validated an autophagy-associated risk model based on MYC, MAPK1, IRGM, PRKCQ, and FOXO1. And those five genes may influence the progression of IPF by regulating immune responses and immune cells.

Telomere-related gene risk model for prognosis and drug treatment efficiency prediction in kidney cancer.

Kidney cancer is one of the most common urological cancers worldwide, and kidney renal clear cell cancer (KIRC) is the major histologic subtype. Our previous study found that von-Hippel Lindau (VHL) gene mutation, the dominant reason for sporadic KIRC and hereditary kidney cancer-VHL syndrome, could affect VHL disease-related cancers development by inducing telomere shortening. However, the prognosis role of telomere-related genes in kidney cancer has not been well discussed. In this study, we obtained the telomere-related genes (TRGs) from TelNet. We obtained the clinical information and TRGs expression status of kidney cancer patients in The Cancer Genome Atlas (TCGA) database, The International Cancer Genome Consortium (ICGC) database, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Totally 353 TRGs were differential between tumor and normal tissues in the TCGA-KIRC dataset. The total TCGA cohort was divided into discovery and validation TCGA cohorts and then using univariate cox regression, lasso regression, and multivariate cox regression method to conduct data analysis sequentially, ten TRGs (ISG15, RFC2, TRIM15, NEK6, PRKCQ, ATP1A1, ELOVL3, TUBB2B, PLCL1, NR1H3) risk model had been constructed finally. The kidney patients in the high TRGs risk group represented a worse outcome in the discovery TCGA cohort (p<0.001), and the result was validated by these four cohorts (validation TCGA cohort, total TCGA cohort, ICGC cohort, and CPTAC cohort). In addition, the TRGs risk score is an independent risk factor for kidney cancer in all these five cohorts. And the high TRGs risk group correlated with worse immune subtypes and higher tumor mutation burden in cancer tissues. In addition, the high TRGs risk group might benefit from receiving immune checkpoint inhibitors and targeted therapy agents. Moreover, the proteins NEK6, RF2, and ISG15 were upregulated in tumors both at the RNA and protein levels, while PLCL1 and PRKCQ were downregulated. The other five genes may display the contrary expression status at the RNA and protein levels. In conclusion, we have constructed a telomere-related genes risk model for predicting the outcomes of kidney cancer patients, and the model may be helpful in selecting treatment agents for kidney cancer patients.

MAP4K3/GLK inhibits Treg differentiation by direct phosphorylating IKKß and inducing IKKß-mediated FoxO1 nuclear export and Foxp3 downregulation.

Rationale: GLK (MAP4K3) activates PKCθ-IKKß axis in T-cell activation and induces IL-17A-mediated autoimmune diseases. Attenuation of Treg differentiation and function by GLK could also contribute to autoimmune diseases. Methods: We analyzed the roles of GLK and IKKß in Treg differentiation and function using T-cell-specific GLK transgenic mice and IKKß conditional knockout mice. The mechanism of GLK/IKKß-mediated attenuation of Treg differentiation/function was studied by chromatin-immunoprecipitation, reporter assays, in vitro kinase assays, protein-protein interaction assays, mass spectrometry, confocal microscopy, flow cytometry, and single-cell RNA sequencing (scRNA-seq) analysis. Results: We found that GLK signaling inhibited Foxp3 transcription by blocking the function of the transcription factor FoxO1. Mechanistically, GLK directly phosphorylated and activated IKKß at Ser733 in a PKCθ-independent manner. The phospho-IKKß Ser733 induced FoxO1 Ser319 phosphorylation and nuclear export, leading to Foxp3 downregulation. Consistently, scRNA-seq analyses showed that Foxp3 mRNA levels were inversely correlated with FoxO1 mRNA levels in GLK transgenic CD4+ T cells. Conclusions: GLK-IKKß-FoxO1 signaling axis inhibits Foxp3 transcription, leading to reduction of Treg differentiation and suppressive activity, as well as induction of autoimmune disease.

Macrophage mitochondrial fission improves cancer cell phagocytosis induced by therapeutic antibodies and is impaired by glutamine competition.

Phagocytosis is required for the optimal efficacy of many approved and promising therapeutic antibodies for various malignancies. However, the factors that determine the response to therapies that rely on phagocytosis remain largely elusive. Here, we demonstrate that mitochondrial fission in macrophages induced by multiple antibodies is essential for phagocytosis of live tumor cells. Tumor cells resistant to phagocytosis inhibit mitochondrial fission of macrophages by overexpressing glutamine-fructose-6-phosphate transaminase 2 (GFPT2), which can be targeted to improve antibody efficacy. Mechanistically, increased cytosolic calcium by mitochondrial fission abrogates the phase transition of the Wiskott-Aldrich syndrome protein (WASP)-Wiskott-Aldrich syndrome interacting protein (WIP) complex and enables protein kinase C-θ (PKC-θ) to phosphorylate WIP during phagocytosis. GFPT2-mediated excessive use of glutamine by tumor cells impairs mitochondrial fission and prevents access of PKC-θ to compartmentalized WIP in macrophages. Our data suggest that mitochondrial dynamics dictate the phase transition of the phagocytic machinery and identify GFPT2 as a potential target to improve antibody therapy.

Crocin attenuates NF-κB-mediated inflammation and proliferation in breast cancer cells by down-regulating PRKCQ.

Breast cancer (BC) is the most commonly diagnosed cancer confronting women worldwide. Crocin, a glycosylated carotenoid extracted from Crocus sativus L., possesses anti-cancer and anti-inflammatory activities. This study tried to explore the influences of crocin on proliferation and inflammation of BC cells, and to investigate the possible mechanism. The protein levels of protein kinase C theta (PRKCQ) and nuclear factor kappa B (NF-κB) p-p65 and p65 were examined using western blot analysis. The potential targets of crocin were predicted using the PharmMapper database. Cell viability and proliferation were determined utilizing CCK-8 and EdU incorporation assays, respectively. Inflammation was assessed by detecting the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) using RT-qPCR and ELISA. Results showed that crocin inhibited NF-κB activation and suppressed cell viability and proliferation in BC cells. Crocin caused a significant reduction of levels of TNF-α and IL-1ß, suggesting that crocin suppressed inflammation in BC cells. NF-κB inhibition decreased proliferation and inflammation in BC cells. Additionally, PRKCQ was identified as a potential target of crocin according to PharmMapper database. Crocin treatment inhibited the activation of NF-κB in BC cells by reducing PRKCQ expression. Mechanistically, PRKCQ-dependent activation of NF-κB pathway reversed the effects of crocin on the proliferation and inflammation in BC cells. In conclusion, crocin inhibited NF-κB-mediated inflammation and proliferation in BC cells through reducing PRKCQ expression.

Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity.

Natural killer (NK) cells play a crucial role in immunity, killing virally infected and cancerous cells. The balance of signals initiated upon engagement of activating and inhibitory NK receptors with cognate ligands determines killing or tolerance. Nevertheless, the molecular mechanisms regulating rapid NK cell discrimination between healthy and malignant cells in a heterogeneous tissue environment are incompletely understood. The SHP-1 tyrosine phosphatase is the central negative NK cell regulator that dephosphorylates key activating signaling proteins. Though the mechanism by which SHP-1 mediates NK cell inhibition has been partially elucidated, the pathways by which SHP-1 is itself regulated remain unclear. Here, we show that phosphorylation of SHP-1 in NK cells on the S591 residue by PKC-θ promotes the inhibited SHP-1 'folded' state. Silencing PKC-θ maintains SHP-1 in the active conformation, reduces NK cell activation and cytotoxicity, and promotes tumor progression in vivo. This study reveals a molecular pathway that sustains the NK cell activation threshold through suppression of SHP-1 activity.

The Pellino1-PKCθ Signaling Axis Is an Essential Target for Improving Antitumor CD8+ T-lymphocyte Function.

CD8+ T cells play an important role in the elimination of tumors. However, the underlying mechanisms involved in eliciting and maintaining effector responses in CD8+ T cells remain to be elucidated. Pellino1 (Peli1) is a receptor signal-responsive ubiquitin E3 ligase, which acts as a critical mediator for innate immunity. Here, we found that the risk of developing tumors was dependent on Peli1 expression. Peli1 was upregulated in CD8+ T cells among tumor-infiltrating lymphocytes (TIL). In contrast, a deficit of Peli1 enhanced the maintenance and effector function of CD8+ TILs. The development of Peli1-deficient CD8+ TILs prevented T-cell exhaustion and retained the hyperactivated states of T cells to eliminate tumors. We also found that Peli1 directly interacted with protein kinase C-theta (PKCθ), a central kinase in T-cell receptor downstream signal transduction, but whose role in tumor immunology remains unknown. Peli1 inhibited the PKCθ pathway by lysine 48-mediated ubiquitination degradation in CD8+ TILs. In summary, the Peli1-PKCθ signaling axis is a common inhibitory mechanism that prevents antitumor CD8+ T-cell function, and thus targeting Peli1 may be a useful therapeutic strategy for improving cytotoxic T-cell activity.

PKCθ-mediated serine/threonine phosphorylations of FAK govern adhesion and protrusion dynamics within the lamellipodia of migrating breast cancer cells.

The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ's specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

PLCγ1/PKCθ Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression.

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.