Therapeutic hypothermia augments the restorative effects of PKC-β and Nox2 inhibition on an in vitro model of human blood–brain barrier

To investigate whether therapeutic hypothermia augments the restorative impact of protein kinase C-β (PKC-β) and Nox2 inhibition on an in vitro model of human blood–brain barrier (BBB). Cells cultured in normoglycaemic (5.5 mM) or hyperglycaemic (25 mM, 6 to 120 h) conditions were treated with therapeutic hypothermia (35 °C) in the absence or presence of a PKC-β inhibitor (LY333531, 0.05 μM) or a Nox2 inhibitor (gp91ds-tat, 50 μM). BBB was established by co-culture of human brain microvascular endothelial cells (HBMECs) with astrocytes (HAs) and pericytes. BBB integrity and function were assessed via transendothelial electrical resistance (TEER) and paracellular flux of sodium fluorescein (NaF, 376 Da). Nox activity (lucigenin assay), superoxide anion production (cytochrome-C reduction assay), cellular proliferative capacity (wound scratch assay) and actin cytoskeletal formation (rhodamine-phalloidin staining) were assessed both in HBMECs and HAs using the specific methodologies indicated in brackets. Therapeutic hypothermia augmented the protective effects of PKC-β or Nox2 inhibition on BBB integrity and function in experimental setting of hyperglycaemia, as evidenced by increases in TEER and concomitant decreases in paracellular flux of NaF. The combinatory approaches were more effective in repairing physical damage exerted on HBMEC and HA monolayers by wound scratch and in decreasing Nox activity and superoxide anion production compared to sole treatment regimen with either agent. Similarly, the combinatory approaches were more effective in suppressing actin stress fibre formation and maintaining normal cytoskeletal structure. Therapeutic hypothermia augments the cerebral barrier-restorative capacity of agents specifically targeting PKC-β or Nox2 pathways.

Protein Kinase C Downregulation upon Rapamycin Treatment Attenuates Neuroinflammation and Mitochondrial Disease

Mitochondrial dysfunction causes many poorly understood diseases, such as Leigh Syndrome, that are often caused by dysfunctions in proteins involved in the electron transport chain. My lab previously reported mTOR is pathologically involved in the neurodegenerative phenotype and premature death of mice missing the Complex I subunit Ndufs4 (Ndufs4-/- mice). We discovered treatment with rapamycin extends lifespan, reduces neuroinflammation, and attenuates the neurodegenerative phenotype in these mice, although the mechanisms remain unclear. Rapamycin-treated Ndufs4-/- mice exhibited decreased activation of the mTORC1 pathway. It also deactivated the mTORC2 pathway. We observed that phosphorylation of the canonical protein kinase C (PKC) isoforms (PKC-α, -β, and -γ) decreased more than any other kinases, leading us to hypothesize its deactivation contributes to the observed lifespan extension. To test this, we treated Ndufs4-/- mice with three different PKC inhibitors: the pan-PKC inhibitors GO6983 and GF109203X, and the PKC-β specific inhibitor ruboxistaurin. Similar to rapamycin, all three drugs were able to significantly delay the onset of neurological symptoms (i.e. clasping) and increase survival. We also observed that PKC-β inhibition reduced skin inflammation to suppress the hair loss phenotype displayed by Ndufs4-/- mice at weaning. We further discovered PKC-β inhibition reduces neuroinflammation by deactivating the NF-kB inflammatory pathway. These results suggest that mTORC2 may play a critical role in the etiology of mitochondrial diseases such as Leigh Syndrome.

Protein Kinase C Isozymes Associated With Relapse Free Survival in Non-Small Cell Lung Cancer Patients

IntroductionProtein expression is deregulated in cancer, and the proteomic changes observed in lung cancer may be a consequence of mutations in essential genes. The purpose of this study was to identify protein expression associated with prognosis in lung cancers stratified by smoking status, molecular subtypes, and EGFR-, TP53-, and KRAS-mutations.MethodsWe performed profiling of 295 cancer-relevant phosphorylated and non-phosphorylated proteins, using reverse phase protein arrays. Biopsies from 80 patients with operable lung adenocarcinomas were analyzed for protein expression and association with relapse free survival (RFS) were studied.ResultsSpearman’s rank correlation analysis identified 46 proteins with significant association to RFS (p<0.05). High expression of protein kinase C (PKC)-α and the phosporylated state of PKC-α, PKC-β, and PKC-δ, showed the strongest positive correlation to RFS, especially in the wild type samples. This was confirmed in gene expression data from 172 samples. Based on protein expression, unsupervised hierarchical clustering separated the samples into four subclusters enriched with the molecular subtypes terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI) (p=0.0001). Subcluster 2 contained a smaller cluster (2a) enriched with samples of the subtype PP, low expression of the PKC isozymes, and associated with poor RFS (p=0.003) compared to the other samples. Low expression of the PKC isozymes in the subtype PP and a reduced relapse free survival was confirmed with The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) samples.ConclusionThis study identified different proteins associated with RFS depending on molecular subtype, smoking- and mutational-status, with PKC-α, PKC-β, and PKC-δ showing the strongest correlation.

Overcoming Multidrug Resistance in B Cell Malignancies By Antagonism of Stromal Mediated TGF-β Signalling

Novel targeted therapies have substantially improved the prognosis of patients with B cell malignancies. However, a substantial fraction of patients still relapse, even after initially achieving deep remissions. Many studies have characterized the interactions between tumor cells and their microenvironment as integral to leukemia/ lymphoma homeostasis and for the provision of survival signals, also contributing to drug resistance (referred to as environment-mediated drug resistance (EMDR)). Therapeutic efforts to antagonize microenvironment-emanating survival cues have predominantly focused on perturbation of tumour cell adhesion enabling the physical displacement from protective niches (e.g. BCR-inhibitors). In an effort to address whether direct stromal targeting could more precisely mitigate EMDR, we recently characterised the molecular mechanisms underlying tumor-stroma interactions in B cell malignancies and identified a protein kinase C-β (PKC-β) as an essential kinase, required for activation of NF-κB in mesenchymal stromal cells (Lutzny et al Cancer Cell 2013). The dependency on stroma PKC-β was uniformly found for acute (ALL) and chronic (CLL, MCL) B cell malignancies. Importantly, our data further demonstrate that targeting stroma PKC-β is of key importance for multi-drug resistance of malignant B cells and can be used for therapeutic interventions (Park et al Science Trans Med 2020). Here we demonstrate novel mechanistic insights into stroma-mediated drug resistance in B cell malignancies. We identified that stroma PKC-β drives a transcriptional program in tumor cells, dependent on the activation of TGF-β and BMP-signaling, which ultimately leads to the stabilisation of BCL-XL. Our data show that BCL-XL expression in tumor cells is associated with SMAD1-induction by cytotoxic therapies, which simultaneously suppress SMAD4 expression. Importantly, SMAD1 expression was strictly dependent on stromal PKC-β activity. Antagonizing stroma signals with TGF-β inhibitors inhibits SMAD1 induction, abrogates the up-regulation of BCL-XL and overcomes stroma-dependent resistance to Venetoclax and conventional chemotherapy. The TGF-β pathway operates in parallel to the activation of the transcription factor EB (TFEB) as a down-stream target of PKC-β. Interference with these signaling pathways impairs plasma membrane integrity of stromal cells by down-regulation of numerous adhesion and signaling molecules, such as ADAM17, required for the reciprocal stabilization of BCL-XL in tumor cells. The significance of microenvironment PKC-β for drug resistance was demonstrated in vivo, using C57B/6 mice, diseased with EμTCL-1 driven B cell tumors and treated with Venetoclax in combination with or without PKC-β inhibitors. Combined treatment significantly prolonged survival, based on PKC-β mediated impairment of EMDR. Similarly, concurrent treatment of PKC-β inhibitors with chemotherapy also improved survival in an ALL-PDx model Our data demonstrate that mitigating EMDR with small molecule inhibitors of PKC-β or TGF-β signalling enhance the effectiveness of both targeted and non-targeted chemotherapies and moreover, has the ability to overcome Venetoclax resistance in B cell malignancies. Clinical trials with repurposed drugs inhibiting the here described pathways mediating EMDR are in planning. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Midostaurin as inhibitor of stroma PKC-β

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II’s ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II’s ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-β, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (−15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (−0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group ( P < 0.046). ANG II did not stimulate PKC-β in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min−1·mg protein−1 in fructose group, and the PKC-α/β inhibitor Gö6976 blocked this increase (−96 ± 205 nmol·min−1·mg protein−1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group ( P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II’s ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Stromal cell protein kinase C-β inhibition enhances chemosensitivity in B cell malignancies and overcomes drug resistance

Overcoming drug resistance remains a key challenge to cure patients with acute and chronic B cell malignancies. Here, we describe a stromal cell–autonomous signaling pathway, which contributes to drug resistance of malignant B cells. We show that protein kinase C (PKC)–β–dependent signals from bone marrow–derived stromal cells markedly decrease the efficacy of cytotoxic therapies. Conversely, small-molecule PKC-β inhibitors antagonize prosurvival signals from stromal cells and sensitize tumor cells to targeted and nontargeted chemotherapy, resulting in enhanced cytotoxicity and prolonged survival in vivo. Mechanistically, stromal PKC-β controls the expression of adhesion and matrix proteins, required for activation of phosphoinositide 3-kinases (PI3Ks) and the extracellular signal–regulated kinase (ERK)–mediated stabilization of B cell lymphoma–extra large (BCL-XL) in tumor cells. Central to the stroma-mediated drug resistance is the PKC-β–dependent activation of transcription factor EB, regulating lysosome biogenesis and plasma membrane integrity. Stroma-directed therapies, enabled by direct inhibition of PKC-β, enhance the effectiveness of many antileukemic therapies.

Protein kinase C isozymes; predictors of progression free survival in NSCLC patients

Background Protein expression is deregulated in cancer, and the proteomic changes observed in lung cancer may be a consequence of mutations in essential genes. The purpose of this study was to identify protein expression associated with prognosis in lung cancers stratified by smoking status, molecular subtypes, and EGFR-, TP53- and KRAS-mutations. Methods We performed profiling of 295 cancer-relevant phosphorylated and non-phosphorylated proteins, using reverse phase protein arrays. Biopsies from 80 patients with operable lung adenocarcinomas were analyzed for protein expression and association with progression free survival (PFS) were studied. Results Spearman rank correlation analysis identified 56 proteins with significant association to PFS (p<0.05). High expression of protein kinase C (PKC)-α and the phosporylated state of PKC-α, PKC-β and PKC-δ, showed the strongest positive correlation to PFS, especially in the wild type samples. This was confirmed in gene expression data from 186 samples. Based on protein expression, unsupervised hierarchical clustering separated the samples into four subclusters enriched with the molecular subtypes TRU, PI or PP (p=0.0001). Subcluster 2 contained a smaller cluster (2a) enriched with samples of the subtype PP, low expression of the PKC isozymes, and associated with poor PFS (p=0.003) compared to the other samples. Subcluster 2a revealed increased expression of neuroendocrine markers, supporting the aggressive behavior. Low expression of the PKC isozymes in the subtype PP and a reduced relapse free survival was confirmed with the TCGA LUAD samples. Conclusion This study identified different proteins associated with PFS depending on molecular subtype, smoking- and mutational-status, with PKC-α, PKC-β and PKC-δ showing the strongest correlation. Cluster analysis detected a subgroup of samples enriched for samples of the PP subtype and poor PFS, which may benefit from a more aggressive treatment regimen.