Atypical Protein Kinase Cs in Melanoma Progression

Melanoma is one of the fastest growing types of cancer worldwide in terms of incidence. To date, reports show over 92,000 new cases in the United States in 2018. Previously, we introduced protein kinase C-iota (PKC-ι) as an oncogene in melanoma. PKC-ι promotes survival and cancer progression along with PKC-zeta(ζ). In addition, we reported that PKC-ι induced metastasis of melanoma cells by increasing Vimentin dynamics. Our previous results showed that PKC-ι inhibition downregulated epithelial-mesenchymal transition (EMT), while inducing apoptosis. In this chapter, we summarized these findings which were based on the in-vitro applications of five specific atypical PKC (aPKC) inhibitors. In addition, the underlying mechanisms of the transcriptional regulation of PRKCI gene expression in melanoma is also discussed. Results demonstrated that c-Jun promotes PRKCI expression along with Interleukin (IL)-6/8. Furthermore, forkhead box protein O1 (FOXO1) acts as a downregulator of PRKCI expression upon stimulation of IL-17E and intercellular adhesion molecule 1 (ICAM-1) in melanoma cells. Overall, the chapter summarizes the importance of PKC-ι/ζ in the progression of melanoma and discusses the cellular signaling pathways that are altered upon inhibitor applications. Finally, we established that aPKCs are effective novel biomarkers for use in the design of novel targeted therapeutics for melanoma

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Oncogenic PKC-ι activates Vimentin during Epithelial-mesenchymal Transition in Melanoma; A Study based on PKC-ι and PKC-ζ specific Inhibitors

Melanoma is one of the fastest growing cancers in the United States and is accompanied with a poor prognosis owing to tumors being resistant to most therapies. Atypical protein kinase Cs (aPKC) are involved in malignancy in many cancers. We previously reported that aPKCs play a key role in melanoma\u27s cell motility by regulating cell signaling pathways which induce epithelial-mesenchymal Transition (EMT). We tested three novel inhibitors; [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) which are specific to protein kinase C-iota (PKC-ι) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) which is specific to PKC-zeta (PKC-ζ) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular modeling was used to identify potential binding sites for the inhibitors and to predict selectivity. Kinase assay showed \u3e50% inhibition for specified targets beyond 5 μM for all inhibitors. Both ICA-1 and ζ-Stat significantly reduced cell proliferation and induced apoptosis, while ICA-1 also significantly reduced migration and melanoma cell invasion. PKC-ι stimulated EMT via TGFβ/Par6/RhoA pathway and activated Vimentin by phosphorylation at S39. Both ICA-1 and ζ-Stat downregulate TNF-α induced NF-κB translocation to the nucleus there by inducing apoptosis. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Inhibitors proved to be effective under in-vitro conditions and need to be tested in-vivo for the validity as effective therapeutics. Overall, results show that aPKCs are essential for melanoma progression and metastasis and that they could be used as effective therapeutic targets for malignant melanoma

C‑Jun and FOXO1 Mediate the Expression of Oncogenic PKC‑ι in Human Prostate Cancer Cells with an Interplay Between NF‑κB, IL‑8 and ICAM‑1

Aggressive and metastatic prostate cancers are among the leading causes of fatality in men. Prior observations by the authors regarding atypical protein kinase C isoforms (aPKCs) in relation to prostate cancers demonstrated elevated levels of PKC‑iota (PKC‑ι) in patient samples compared to non‑malignant prostate tissues. This indicates that PKC‑ι is a potential biomarker for initiating and maintaining prostate carcinogenesis. In addition, PKC‑ι is an oncogene that encourages the activation of the nuclear factor (NF)‑κB, assisting carcinogenesis. The specific inhibition of PKC‑ι de‑regulated the expression of both PKC‑ι and its phosphorylation; thus, PKC‑ι functionally controls its own expression in prostate carcinoma. The present study aimed to investigate the underlying mechanisms of PRKCI gene transcriptional regulation in prostate carcinoma cells. Forkhead box protein O1 (FOXO1) and c‑Jun, along with several other transcription factors that exhibited potential to bind on or near the promoter region of the PRKCI were identified. Each transcription factor was systematically silenced. The decrease in the expression levels of FOXO1 and c‑Jun significantly affected PKC‑ι expression. The decrease in FOXO1 expression by siRNA enhanced PKC‑ι expression by 33% (P≤0.05) and 9% (P≤0.05) in the PC‑3 and DU‑145 cells, respectively. The diminution of c‑Jun expression by siRNA diminished PKC‑ι expression by 42% (P≤0.05) and 24% (P≤0.05) in the PC‑3 and DU‑145 cells, respectively. According to the results of the present study, c‑Jun and FOXO1 are the two major transcription factors involved in PKC‑ι expression in prostate cells. PKC‑ι and its phosphorylation improved due to FOXO1 diminution, and vice versa for c‑Jun silencing, indicating that c‑Jun upregulates PRKCI expression, while FOXO1 negatively affects its expression. This was mediated through signal transducer and activator of transcription (STAT)3/5 and NF‑κB. An upregulation in the expression of intercellular adhesion molecule 1 (ICAM‑1) and interleukin (IL)‑8 was observed as a result of PKC‑ι specific inhibition. PKC‑ι inhibition thus promotes ICAM‑1/FOXO1 signaling and downregulates IL‑8/JNK/c‑Jun signaling, indicating that PKC‑ι inhibition subdues its production mechanism. Overall, an analysis of the results led us to suggest that PKC‑ι inhibition downregulates its own oncogenic signaling, while the induction of anti‑tumor signaling pathways strongly suggests that PKC‑ι related molecular mechanisms provide a novel therapeutic route for mitigating prostate cancer

C‑Jun and FOXO1 Mediate the Expression of Oncogenic PKC‑ι in Human Prostate Cancer Cells with an Interplay Between NF‑κB, IL‑8 and ICAM‑1

Aggressive and metastatic prostate cancers are among the leading causes of fatality in men. Prior observations by the authors regarding atypical protein kinase C isoforms (aPKCs) in relation to prostate cancers demonstrated elevated levels of PKC‑iota (PKC‑ι) in patient samples compared to non‑malignant prostate tissues. This indicates that PKC‑ι is a potential biomarker for initiating and maintaining prostate carcinogenesis. In addition, PKC‑ι is an oncogene that encourages the activation of the nuclear factor (NF)‑κB, assisting carcinogenesis. The specific inhibition of PKC‑ι de‑regulated the expression of both PKC‑ι and its phosphorylation; thus, PKC‑ι functionally controls its own expression in prostate carcinoma. The present study aimed to investigate the underlying mechanisms of PRKCI gene transcriptional regulation in prostate carcinoma cells. Forkhead box protein O1 (FOXO1) and c‑Jun, along with several other transcription factors that exhibited potential to bind on or near the promoter region of the PRKCI were identified. Each transcription factor was systematically silenced. The decrease in the expression levels of FOXO1 and c‑Jun significantly affected PKC‑ι expression. The decrease in FOXO1 expression by siRNA enhanced PKC‑ι expression by 33% (P≤0.05) and 9% (P≤0.05) in the PC‑3 and DU‑145 cells, respectively. The diminution of c‑Jun expression by siRNA diminished PKC‑ι expression by 42% (P≤0.05) and 24% (P≤0.05) in the PC‑3 and DU‑145 cells, respectively. According to the results of the present study, c‑Jun and FOXO1 are the two major transcription factors involved in PKC‑ι expression in prostate cells. PKC‑ι and its phosphorylation improved due to FOXO1 diminution, and vice versa for c‑Jun silencing, indicating that c‑Jun upregulates PRKCI expression, while FOXO1 negatively affects its expression. This was mediated through signal transducer and activator of transcription (STAT)3/5 and NF‑κB. An upregulation in the expression of intercellular adhesion molecule 1 (ICAM‑1) and interleukin (IL)‑8 was observed as a result of PKC‑ι specific inhibition. PKC‑ι inhibition thus promotes ICAM‑1/FOXO1 signaling and downregulates IL‑8/JNK/c‑Jun signaling, indicating that PKC‑ι inhibition subdues its production mechanism. Overall, an analysis of the results led us to suggest that PKC‑ι inhibition downregulates its own oncogenic signaling, while the induction of anti‑tumor signaling pathways strongly suggests that PKC‑ι related molecular mechanisms provide a novel therapeutic route for mitigating prostate cancer

Inhibition of Atypical Protein Kinase C‑ι Effectively Reduces the Malignancy of Prostate Cancer cells by Downregulating the NF-κB Signaling Cascade

Prostate cancer (PC) is the most common type of cancer among men. Aggressive and metastatic PC results in lifethreatening tumors, and represents one of the leading causes of mortality in men. Previous studies of atypical protein kinase C isoforms (aPKCs) have highlighted its role in the survival of cultured prostate cells via the nuclear factor (NF)-κB pathway. The present study showed that PKC-ι was overexpressed in PC samples collected from cancer patients but not in non-invasive prostate tissues, indicating PKC-ι as a possible prognostic biomarker for the progression of prostate carcinogenesis. Immunohistochemical staining further confirmed the association between PKC-ι and the prostate malignancy. The DU-145 and PC-3 PC cell lines, and the non-neoplastic RWPE-1 prostatic epithelial cell line were cultured and treated with aPKC inhibitors 2-acetyl-1,3-cyclopentanedione (ACPD) and 5-amino-1-(1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1). Western blot data demonstrated that ICA‑1 was an effective and specific inhibitor of PKC‑ι and that ACPD inhibited PKC-ι and PKC-ζ. Furthermore, the two inhibitors significantly decreased malignant cell proliferation and induced apoptosis. The inhibitors showed no significant cytotoxicity towards the RWPE-1 cells, but exhibited cytostatic effects on the DU-145 and PC-3 cells prior to inducing apoptosis. The inhibition of aPKCs significantly reduced the translocation of NF-κB to the nucleus. Furthermore, this inhibition promoted apoptosis, reduced signaling for cell survival, and reduced the proliferation of PC cells, whereas the normal prostate epithelial cells were relatively unaffected. Overall, the results suggested that PKC-ι and PKC-ζ are essential for the progression of PC, and that ACPD and ICA-1 can be effectively used as potential inhibitors in targeted therapy

Preclinical Testing of Chronic ICA-1S Exposure: A Potent Protein Kinase C-ι Inhibitor as a Potential Carcinoma Therapeutic

Protein kinase C-iota (PKC-ι) is an oncogene overexpressed in many cancer cells including prostate, breast, ovarian, melanoma, and glioma cells. Previous in vitro studies have shown that 5-amino-1-((1R,2S,3R,4R)-2-3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), a PKC-ι-specific inhibitor, has low toxicity in both acute and sub-acute mouse model toxicological testing and is an effective therapeutic against several cancer cell lines showing significant reductions in tumor growth when treating athymic nude mice with xenografted carcinoma cell lines. To further assess ICA-1S as a possible therapeutic agent, chronic mouse model toxicological testing was performed in vivo to provide inferences concerning the long-term effects and possible health hazards from repeated exposure over a substantial part of the animal’s lifespan. Subjects survived well after 30, 60, and 90 days of doses ranging from 50 mg/kg to 100 mg/kg. Heart, liver, kidney, and brain tissues were then analyzed for accumulations of ICA-1S including the measured assessment of aspartate transaminase (AST), alkaline phosphatase (ALK-P), gamma-glutamyl transferase (GGT), troponin, and C-reactive protein (CRP) serum levels to assess organ function. Predictive in vitro/in silico methods were used to predict compound-induced direct hepatocyte toxicity or renal proximal tubular cell (PTC) toxicity in humans based on the high-content imaging (HCI) of compound-treated cells in combination with phenotypic profiling. In conclusion, ICA-1S shows low toxicity in both acute and chronic toxicology studies, and shows promise as a potential therapeutic