Protein kinase C signaling "in" and "to" the nucleus: Master kinases in transcriptional regulation.

PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases.

∆Np63α inhibits Rac1 activation and cancer cell invasion through suppression of PREX1.

ΔNp63α, a member of the p53 family of transcription factors, plays a critical role in maintaining the proliferative potential of stem cells in the stratified epithelium. Although ΔNp63α is considered an oncogene and is frequently overexpressed in squamous cell carcinoma, loss of ΔNp63α expression is associated with increased tumor cell invasion and metastasis. We recently identified a ΔNp63α/miR-320a/PKCγ signaling axis that regulates cancer cell invasion by inhibiting phosphorylation of the small GTPase Rac1, a master switch of cell motility that positively regulates cell invasion in multiple human cancers. In this study, we identified a novel mechanism by which ΔNp63α negatively regulates Rac1 activity, by inhibiting the expression of the Rac-specific Guanine Exchange Factor PREX1. ΔNp63α knockdown in multiple squamous cell carcinoma cell lines leads to increased Rac1 activation, which is abrogated by treatment with the Rac1 inhibitor NSC23766. Furthermore, ΔNp63α negatively regulates PREX1 transcript and protein levels. Using a Rac-GEF activation assay, we also showed that ΔNp63α reduces the levels of active PREX1. The inhibition of the PREX1-Rac1 signaling axis by ΔNp63α leads to impaired cell invasion, thus establishing the functional relevance of this link. Our results elucidated a novel molecular mechanism by which ΔNp63α negatively affects cancer cell invasion and identifies the ΔNp63α/Rac1 axis as a potential target for metastasis.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gßγ/PKCα/FARP1/Cdc42 axis.

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Germline intergenic duplications at Xq26.1 underlie Bazex-Dupré-Christol basal cell carcinoma susceptibility syndrome.

BACKGROUND: Bazex-Dupré-Christol syndrome (BDCS; MIM301845) is a rare X-linked dominant genodermatosis characterized by follicular atrophoderma, congenital hypotrichosis and multiple basal cell carcinomas (BCCs). Previous studies have linked BDCS to an 11·4-Mb interval on chromosome Xq25-q27.1. However, the genetic mechanism of BDCS remains an open question. OBJECTIVES: To investigate the genetic aetiology and molecular mechanisms underlying BDCS. METHODS: We ascertained multiple individuals from eight unrelated families affected with BDCS (F1-F8). Whole-exome (F1 and F2) and genome sequencing (F3) were performed to identify putative disease-causing variants within the linkage region. Array comparative genomic hybridization and quantitative polymerase chain reaction (PCR) were used to explore copy number variations, followed by long-range gap PCR and Sanger sequencing to amplify the duplication junctions and to define the head-tail junctions. Hi-C was performed on dermal fibroblasts from two affected individuals with BDCS and one control. Public datasets and tools were used to identify regulatory elements and transcription factor binding sites within the minimal duplicated region. Immunofluorescence was performed in hair follicles, BCCs and trichoepitheliomas from patients with BDCS and sporadic BCCs. The ACTRT1 variant c.547dup (p.Met183Asnfs*17), previously proposed to cause BDCS, was evaluated with t allele frequency calculator. RESULTS: In eight families with BDCS, we identified overlapping 18-135-kb duplications (six inherited and two de novo) at Xq26.1, flanked by ARHGAP36 and IGSF1. Hi-C showed that the duplications did not affect the topologically associated domain, but may alter the interactions between flanking genes and putative enhancers located in the minimal duplicated region. We detected ARHGAP36 expression near the control hair follicular stem cell compartment, and found increased ARHGAP36 levels in hair follicles in telogen, in BCCs and in trichoepitheliomas from patients with BDCS. ARHGAP36 was also detected in sporadic BCCs from individuals without BDCS. Our modelling showed the predicted maximum tolerated minor allele frequency of ACTRT1 variants in control populations to be orders of magnitude higher than expected for a high-penetrant ultra-rare disorder, suggesting loss of function of ACTRT1 variants to be an unlikely cause for BDCS. CONCLUSIONS: Noncoding Xq26.1 duplications cause BDCS. The BDCS duplications most likely lead to dysregulation of ARHGAP36. ARHGAP36 is a potential therapeutic target for both inherited and sporadic BCCs. What is already known about this topic? Bazex-Dupré-Christol syndrome (BDCS) is a rare X-linked basal cell carcinoma susceptibility syndrome linked to an 11·4-Mb interval on chromosome Xq25-q27.1. Loss-of-function variants in ACTRT1 and its regulatory elements were suggested to cause BDCS. What does this study add? BDCS is caused by small tandem noncoding intergenic duplications at chromosome Xq26.1. The Xq26.1 BDCS duplications likely dysregulate ARHGAP36, the flanking centromeric gene. ACTRT1 loss-of-function variants are unlikely to cause BDCS. What is the translational message? This study provides the basis for accurate genetic testing for BDCS, which will aid precise diagnosis and appropriate surveillance and clinical management. ARHGAP36 may be a novel therapeutic target for all forms of sporadic basal cell carcinomas.

Quantification of ruffle area and dynamics in live or fixed lung adenocarcinoma cells.

Ruffles are actin-rich membrane protrusions implicated in actin reorganization and initiation of cell motility. Here, we describe methods for measuring and analyzing ruffle dynamics in live cells and average ruffle area per cell in fixed samples. The specific steps described are for the analysis of A549 lung adenocarcinoma cells, but the protocol can be applied to other cell types. The protocol has applications for dissecting the signaling events linked to ruffling. For complete details on the use and execution of this protocol, please refer to Cooke et al. (2021).

Nonredundant Rac-GEF control of actin cytoskeleton reorganization.

Rac-GEFs operate in a nonredundant manner as downstream effectors of receptor tyrosine kinases to promote ruffle formation, indicative of unique modes of regulation and targeting. Current research is shedding light on the intricate signaling paradigms shaping spatiotemporal activation of the small GTPase Rac during the generation of actin-rich membrane protrusions.

Overarching roles of diacylglycerol signaling in cancer development and antitumor immunity.

Diacylglycerol (DAG) is a lipid second messenger that is generated in response to extracellular stimuli and channels intracellular signals that affect mammalian cell proliferation, survival, and motility. DAG exerts a myriad of biological functions through protein kinase C (PKC) and other effectors, such as protein kinase D (PKD) isozymes and small GTPase-regulating proteins (such as RasGRPs). Imbalances in the fine-tuned homeostasis between DAG generation by phospholipase C (PLC) enzymes and termination by DAG kinases (DGKs), as well as dysregulation in the activity or abundance of DAG effectors, have been widely associated with tumor initiation, progression, and metastasis. DAG is also a key orchestrator of T cell function and thus plays a major role in tumor immunosurveillance. In addition, DAG pathways shape the tumor ecosystem by arbitrating the complex, dynamic interaction between cancer cells and the immune landscape, hence representing powerful modifiers of immune checkpoint and adoptive T cell-directed immunotherapy. Exploiting the wide spectrum of DAG signals from an integrated perspective could underscore meaningful advances in targeted cancer therapy.

Activating RAC1 variants in the switch II region cause a developmental syndrome and alter neuronal morphology.

RAC1 is a highly conserved Rho GTPase critical for many cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Some of these variants have previously been shown to have a dominant negative effect. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. Here, we describe eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblast spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborization neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause a developmental syndrome. Our findings reveal that these variants cause altered downstream signalling, resulting in abnormal neuronal morphology and reveal the WAVE regulatory complex/Arp2/3 pathway as a possible therapeutic target for activating RAC1 variants. These insights also have the potential to inform the mechanism and therapy for other disorders caused by variants in genes encoding other Rho GTPases, their regulators and downstream effectors.

The anti-Rac1-GTP antibody and the detection of active Rac1: a tool with a fundamental flaw.

Rac1 is a member of the Rho GTPase family and is involved in many cellular processes, particularly the formation of actin-rich membrane protrusions, such as lamellipodia and ruffles. With such a widely studied protein, it is essential that the research community has reliable tools for detecting Rac1 activation both in cellular models and tissues. Using a series of cancer cellular models, we recently demonstrated that a widely used antibody for visualizing active Rac1 (Rac1-GTP) does not recognize Rac1 but instead recognizes vimentin filaments (Baker MJ, J. Biol. Chem. 295:13698-13710, 2020). We believe that this tool has misled the field and impose on the GTPase research community the need to validate published results using this antibody as well as to continue the development of new resources to visualize endogenous active Rac1.

PROTEIN KINASE C ALPHA IS A CENTRAL NODE FOR TUMORIGENIC TRANSCRIPTIONAL NETWORKS IN HUMAN PROSTATE CANCER.

Aberrant expression of protein kinase C (PKC) isozymes is a hallmark of cancer. The different members of the PKC family control cellular events associated with cancer development and progression. Whereas the classical/conventional PKCα isozyme has been linked to tumor suppression in most cancer types, here we demonstrate that this kinase is required for the mitogenic activity of aggressive human prostate cancer cells displaying aberrantly high PKCα expression. Immunohistochemical analysis showed abnormal up-regulation of PKCα in human primary prostate tumors. Interestingly, silencing PKCα expression from aggressive prostate cancer cells impairs cell cycle progression, proliferation and invasion, as well as their tumorigenic activity in a mouse xenograft model. Mechanistic analysis revealed that PKCα exerts a profound control of gene expression, particularly over genes and transcriptional networks associated with cell cycle progression and E2F transcription factors. PKCα RNAi depletion from PC3 prostate cancer cells led to a reduction in the expression of pro-inflammatory cytokine and epithelial-to-mesenchymal transition (EMT) genes, as well as a prominent down-regulation of the immune checkpoint ligand PD-L1. This PKCα-dependent gene expression profile was corroborated in silico using human prostate cancer databases. Our studies established PKCα as a multifunctional kinase that plays pleiotropic roles in prostate cancer, particularly by controlling genetic networks associated with tumor growth and progression. The identification of PKCα as a pro-tumorigenic kinase in human prostate cancer provides strong rationale for the development of therapeutic approaches towards targeting PKCα or its effectors.

Characterization of Novel Derivatives of MBQ-167, an inhibitor of the GTP-binding proteins Rac/Cdc42.

Rac and Cdc42, are homologous GTPases that regulate cell migration, invasion, and cell cycle progression; thus, representing key targets for metastasis therapy. We previously reported on the efficacy of MBQ-167, which blocks both Rac1 and Cdc42 in breast cancer cells and mouse models of metastasis. To identify compounds with increased activity, a panel of MBQ-167 derivatives was synthesized, maintaining its 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole core. Similar to MBQ-167, MBQ-168 and EHop-097, inhibit activation of Rac and Rac1B splice variant and breast cancer cell viability, and induce apoptosis. MBQ-167 and MBQ-168 inhibit Rac and Cdc42 by interfering with guanine nucleotide binding, and MBQ-168 is a more effective inhibitor of PAK (1,2,3) activation. EHop-097 acts via a different mechanism by inhibiting the interaction of the guanine nucleotide exchange factor (GEF) Vav with Rac. MBQ-168 and EHop-097 inhibit metastatic breast cancer cell migration, and MBQ-168 promotes loss of cancer cell polarity to result in disorganization of the actin cytoskeleton and detachment from the substratum. In lung cancer cells, MBQ-168 is more effective than MBQ-167 or EHop-097 at reducing ruffle formation in response to EGF. Comparable to MBQ-167, MBQ-168 significantly inhibits HER2+ tumor growth and metastasis to lung, liver, and spleen. Both MBQ-167 and MBQ-168 inhibit the cytochrome P450 (CYP) enzymes 3A4, 2C9, and 2C19. However, MBQ-168 is ~10X less potent than MBQ-167 at inhibiting CYP3A4, thus demonstrating its utility in relevant combination therapies. In conclusion, the MBQ-167 derivatives MBQ-168 and EHop-097 are additional promising anti metastatic cancer compounds with similar and distinct mechanisms.

FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma.

Despite the undisputable role of the small GTPase Rac1 in the regulation of actin cytoskeleton reorganization, the Rac guanine-nucleotide exchange factors (Rac-GEFs) involved in Rac1-mediated motility and invasion in human lung adenocarcinoma cells remain largely unknown. Here, we identify FARP1, ARHGEF39, and TIAM2 as essential Rac-GEFs responsible for Rac1-mediated lung cancer cell migration upon EGFR and c-Met activation. Noteworthily, these Rac-GEFs operate in a non-redundant manner by controlling distinctive aspects of ruffle dynamics formation. Mechanistic analysis reveals a leading role of the AXL-Gab1-PI3K axis in conferring pro-motility traits downstream of EGFR. Along with the positive association between the overexpression of Rac-GEFs and poor lung adenocarcinoma patient survival, we show that FARP1 and ARHGEF39 are upregulated in EpCam+ cells sorted from primary human lung adenocarcinomas. Overall, our study reveals fundamental insights into the complex intricacies underlying Rac-GEF-mediated cancer cell motility signaling, hence underscoring promising targets for metastatic lung cancer therapy.

Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition.

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35-0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type-dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic DAG-Lactones as PKC Activators.

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile group susceptible to endogenous esterases. An interesting challenge involves replacing the ester group of these ligands while still maintaining biological activity. Here, we present the synthesis and functional characterization of novel diacylglycerol-lactones containing heterocyclic ring substituents at the sn-1 position. Our results showed that the new compound 10B12, a DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a novel structural approach to achieve PKC isozyme selectivity.

Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition.

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35 - 0.74], p = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner. Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks. SIGNIFICANCE: COVID-19 is taking a major toll on personal health, healthcare systems, and the global economy. With three betacoronavirus epidemics in less than 20 years, there is an urgent need for therapies to combat new and existing coronavirus outbreaks. Our analysis of clinical data from over 300,000 patients in three major health systems demonstrates a 50% reduced risk of COVID-19 in patients taking lithium, a direct inhibitor of glycogen synthase kinase-3 (GSK-3). We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells. These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future.

PKCε regulates Rho GTPases and actin cytoskeleton reorganization in non-small cell lung cancer cells.

Oncogenic protein kinase C epsilon (PKCε) promotes the formation of membrane ruffles and motility in non-small cell lung cancer (NSCLC) cells. We found that PKCε is down-regulated when NSCLC cells undergo epithelial-to-mesenchymal transition (EMT) in response to TGF-ß, thus becoming dispensable for migration and invasion in the mesenchymal state. PKCε silencing or inhibition leads to stress fibre formation, suggesting that this kinase negatively regulates RhoA activity. Ruffle formation induced by PKCε activation in the epithelial state is dependent on PI3K, but does not involve the PI3K-dependent Rac-GEFs Ect2, Trio, Vav2 or Tiam1, suggesting alternative Rac-GEFs as mediators of this response. In the proposed model, PKCε acts as a rheostat for Rho GTPases that differs in the epithelial and mesenchymal states.

The PKC universe keeps expanding: From cancer initiation to metastasis.

Classical and novel protein kinase C (PKC) isozymes (c/nPKCs), members of the PKC family that become activated by the lipid second messenger diacylglycerol (DAG) and phorbol esters, exert a myriad of cellular effects that impact proliferative and motile cellular responses. While c/nPKCs have been indisputably associated with tumor promotion, their roles exceed by far their sole involvement as promoter kinases. Indeed, this original dogma has been subsequently redefined by the introduction of several new concepts: the identification of tumor suppressing roles for c/nPKCs, and their participation in early and late stages of carcinogenesis. This review dives deep into the intricate roles of c/nPKCs in cancer initiation as well as in the different stages of the metastatic cascade, with great emphasis in their involvement in cancer cell motility via regulation of small Rho GTPases, the production of extracellular matrix (ECM)-degrading proteases, and the epithelial-to-mesenchymal transition (EMT) program required for the acquisition of highly invasive traits. Here, we highlight functional interplays between either PKCα or PKCε and mesenchymal features that may ultimately contribute to anticancer drug resistance in cellular and animal models. We also introduce the novel hypothesis that c/nPKCs may be implicated in the control of immune evasion through the regulation of immune checkpoint protein expression. In summary, dissecting the colossal complexity of c/nPKC signaling in the wide spectrum of cancer progression may bring new opportunities for the development of meaningful tools aiding for cancer prognosis and therapy.

PKCε Is Required for KRAS-Driven Lung Tumorigenesis.

Non-small cell lung cancer (NSCLC) is the most frequent subtype of lung cancer and remains a highly lethal malignancy and one of the leading causes of cancer-related deaths worldwide. Mutant KRAS is the prevailing oncogenic driver of lung adenocarcinoma, the most common histologic form of NSCLC. In this study, we examined the role of PKCϵ, an oncogenic kinase highly expressed in NSCLC and other cancers, in KRAS-driven tumorigenesis. Database analysis revealed an association between PKCϵ expression and poor outcome in patients with lung adenocarcinoma specifically harboring KRAS mutations. A PKCϵ-deficient, conditionally activatable allele of oncogenic Kras (LSL-KrasG12D ;PKCϵ-/- mice) demonstrated the requirement of PKCϵ for Kras-driven lung tumorigenesis in vivo, which was consistent with impaired transformed growth reported in PKCϵ-deficient KRAS-dependent NSCLC cells. Moreover, PKCϵ-knockout mice were found to be less susceptible to lung tumorigenesis induced by benzo[a]pyrene, a carcinogen that induces mutations in Kras. Mechanistic analysis using RNA sequencing revealed little overlap for PKCϵ and KRAS in the control of genes and biological pathways relevant in NSCLC, suggesting that a permissive role of PKCϵ in KRAS-driven lung tumorigenesis may involve nonredundant mechanisms. Our results thus, highlight the relevance and potential of targeting PKCϵ for lung cancer therapeutics. SIGNIFICANCE: These findings demonstrate that KRAS-mediated tumorigenesis requires PKCϵ expression and highlight the potential for developing PKCϵ-targeted therapies for oncogenic RAS-driven malignancies.