André H. Apostolatos

Two novel atypical PKC inhibitors; ACPD and DNDA effectively mitigate cell proliferation and epithelial to mesenchymal transition of metastatic melanoma while inducing apoptosis

Atypical protein kinase Cs (aPKC) are involved in cell cycle progression, tumorigenesis, cell survival and migration in many cancers. We believe that aPKCs play an important role in cell motility of melanoma by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). We have investigated the effects of two novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocytes. Molecular docking data suggested that both inhibitors specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) and kinase activity assays were carried out to confirm these observations. Both inhibitors decreased the levels of total and phosphorylated PKC-ζ and PKC-ι. Increased levels of E-cadherin, RhoA, PTEN and decreased levels of phosphorylated vimentin, total vimentin, CD44, β-catenin and phosphorylated AKT in inhibitor treated cells. This suggests that inhibition of both PKC-ζ and PKC-ι using ACPD and DNDA downregulates EMT and induces apoptosis in melanoma cells. We also carried out PKC-ι and PKC-ζ directed siRNA treatments to prove the above observations. Immunoprecipitation data suggested an association between PKC-ι and vimentin and PKC-ι siRNA treatments confirmed that PKC-ι activates vimentin by phosphorylation. These results further suggested that PKC-ι is involved in signaling pathways which upregulate EMT and which can be effectively suppressed using ACPD and DNDA. Our results summarize that melanoma cells proliferate via aPKC/AKT/NF-κB mediated pathway while inducing the EMT via PKC-ι/Par6/RhoA pathway. Overall, results show that aPKCs are essential for melanoma progression and metastasis, suggesting that ACPD and DNDA can be effectively used as potential therapeutic drugs for melanoma by inhibiting aPKCs.

Abstract 2369: Transcription activators that regulate PKC-iota expression and are downstream targets of PKC-iota

Protein Kinase C-iota (PKC-iota) is an anti-apoptotic oncogene over-expressed in multiple cancers including prostate, ovarian, and glioma. PKC-iota is part of a cycle that helps cancer cell avoid senescence by releasing the transcription factor NFkB and promoting apoptotic resistance. PKC-iota is activated externally by factors like loss of PTEN (Paget 2012). However, while under the effect PKC-iota specific inhibitors, expression levels decreased, suggesting PKC-iota plays a role in regulating its own expression. A previous study showed the ELK1 transcription factor to be a regulator of PKC-iota (Gustafson 2003). Other transcription factors including Jun, ISGF3, PAX3, EGR1, and FOXO1 bind on or near the promoter sequence of the gene and their role in PKC-iota regulation was analyzed. Each transcription factor was systematically silenced with its own siRNA. Western Blotting revealed expression of PKC-iota in the transcription factor silenced cells determining which transcription factors are key players in regulation of PKC-iota. qPCR and microarray were performed to analyze the transcriptome of treated cells to match protein levels with mRNA levels. Targets both up and downstream of PKC-iota were analyzed to find the pathway that PKC-iota uses to help regulate itself. Citation Format: Andre H. Apostolatos, Wishrawana S. Ratnayake, Tracess Smalley, Anisul Islam, Mildred Acevedo-Duncan. Transcription activators that regulate PKC-iota expression and are downstream targets of PKC-iota [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2369. doi:10.1158/1538-7445.AM2017-2369

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 life- threatening 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.

Oncogenic PKC-ι activates Vimentin during epithelial-mesenchymal transition in melanoma; a study based on PKC-ι and PKC-ζ specific inhibitors

ABSTRACT 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 melanomas 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 >50% 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.

Abstract 179: Atypical protein kinase C inhibition in prostate cancer cells: a study of ICA-1 and ACPD

Aggressive and metastatic prostate cancers are highly lethal tumors and are one of the leading causes of death among men. Certain prostate cancers are highly lethal tumors due to the emergence of therapy-resistant cancer cells. There is a gap in knowledge relating to which intracellular chemicals confer the emergence of therapy-resistant prostate cancer cells, which intracellular chemicals are responsible for prostate cancer survival and which chemicals regulate intracellular signaling pathways in prostate cancer. To address this critical problem, we investigated two drugs that act as inhibitors of atypical protein kinase C. The objective of this study was to test the effectiveness of ACPD [Diabetes. 63:2690-2701 (2014)] as an atypical PKC inhibitor and ICA-1 as an inhibitor of PKC-iota [The Inter. J. Biochem. & Cell Biol. 43:784-794(2011)]. DU-145 prostate carcinoma cells and non-malignant prostate RWPE-1 cells were each cultivated in separate flasks. They were treated with drugs of interest for three consecutive days. The cells were counted before and after the treatments and a statistical analysis was performed. The cells were lysed and levels of PKC-iota and PKC-zeta were measured by Western blotting and immunoprecipitation. Our preliminary results demonstrated that treatment of DU-145 showed a statistically significant decrease in cells that was proportional to the concentration in both drugs. Treatment of the RWPE-1 cells showed no statistically significant change in population. Western blotting showed that both drugs decreased PKC-iota. In conclusion ACPD is an effective inhibitor of PKC-iota and PKC-zeta and consequently significantly reduced the neogenesis of the prostate DU-145 carcinoma cells while having negligible effect on the non-malignant prostate RWPE-1 cells. Citation Format: Andre H. Apostolatos, Christopher Apostolatos, Mildred Acevedo-Duncan. Atypical protein kinase C inhibition in prostate cancer cells: A study of ICA-1 and ACPD. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3470. doi:10.1158/1538-7445.AM2015-3470