Dual inhibition of atypical PKC signaling and PI3K/Akt signaling dysregulates c-Myc to induce apoptosis in clear cell Renal Cell Carcinoma.

Background: Renal Cell Carcinoma (RCC) is the most common type of kidney cancer (85%). 75% of the RCC cases involve conventional clear cell RCC (ccRCC). Approximately, 39% of late-stage patients (stage IV) are treated with chemotherapeutic agents. Phosphatidylinositol-3-kinase (PI3K) and Mitogen-Activated Protein Kinase Kinase (MEK)/extracellular signal-regulated kinase (ERK1/2) pathways are frequently activated in RCC. In addition, atypical PKCs (PKC-ί and PKC ζ) are overexpressed in most cancer cells, and they play a central role in tumor progression and the metastasis of different types of cancers. Our goal is to establish the role of aPKCs in the regulation of multiple key activated pathways in ccRCC. In this study, we also established a novel therapeutic regimen for dual inhibition of key activated pathways. Method: In this study, 786-0 and Caki-1 cells were studied and subjected to cell viability assay, western blot analysis, scratch & wound healing assay, transwell invasion assay, immunofluorescence, immunoprecipitation, flow cytometry, and quantitative real-time polymerase chain reaction. We used combination of PI3K inhibitor- Alpelisib (BYL719) and ICA-1 (a PKC-ι-specific 5-amino-1-2,3-dihydroxy-4-(methylcyclopentyl)-1H-imidazole-4-carboxamide). In addition to drug treatment, small interfering RNA (siRNA) technology was used to further confirm the experimental outcome of the drug treatment. Results: Our results suggest that treatment of ccRCC cells with a combination of ICA-1 (aPKC inhibitor) and BYL719 (PI3K inhibitor) downregulates PKC-ί and causes downstream inhibition of c-Myc. Inhibition of the PKCί also reduces activation of MEK/ERK1/2. It is observed that treatment with ICA-1 disrupts the level of the aPKC-Akt1 association. ICA-1 treatment also shows a reduced level of association between aPKC and c-Myc. The inhibition of aPKCs and downstream effector proteins by combination therapy is more pronounced compared to a single therapy. These effects contribute to reduced cell growth, and eventually, the induction of apoptosis. The decreased level of N-cadherin, p-vimentin, and vimentin and the increased level of E-cadherin confirm reduced malignancy. Conclusion: Therefore, implementing a combination of Alpelisib and a PKC-ι inhibitor is an effective approach to reducing cell proliferation, and invasion that eventually induces apoptosis and may be considered as a potential therapeutic option in ccRCC.

Atypical PKCs activate Vimentin to facilitate prostate cancer cell motility and invasion.

Atypical protein kinase C (aPKC) are involved in progression of many human cancers. Vimentin is expressed during epithelial to mesenchymal transition (EMT). Molecular dynamics of Vimentin intermediate filaments (VIFs) play a key role in metastasis. This article is an effort to provide thorough understanding of the relationship between Vimentin and aPKCs . We demonstrate that diminution of aPKCs lead to attenuate prostate cellular metastasis through the downregulation of Vimentin expression. siRNA knocked-down SNAIL1 and PRRX1 reduce aPKC activity along with Vimentin. Results suggest that aPKCs target multiple activation sites (Ser33/39/56) on Vimentin and therefore is essential for VIF dynamics regulation during the metastasis of prostate cancer cells. Understanding the aPKC related molecular mechanisms may provide a novel therapeutic path for prostate carcinoma.

Preclinical testing of 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide: a potent protein kinase C-ι inhibitor as a potential prostate carcinoma therapeutic.

Protein kinase C-iota (PKC-ι) is an oncogene overexpressed in many cancer cells including prostate, breast, ovarian, melanoma, and glioma. Previous in-vitro studies have shown that 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1s), a PKC-ι specific inhibitor, is effective against some cancer cell lines by decreasing cell growth and inducing apoptosis. To assess ICA-1s as a possible therapeutic, in-vivo studies using a murine model were performed. ICA-1s was tested for stability in blood serum and results demonstrated that ICA-1s was stable in human plasma at 25 and 37°C over a course of 2 h. Toxicity of ICA-1s was tested for both acute and subacute exposure. The acute exposure showed patient surviving after 48 h of doses ranging from 5 to 5000 mg/kg. Subacute tests exposed the patients to 14 days of treatment and were followed by serum and tissue collection. Aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, troponin, and C-reactive protein serum levels were measured to assess organ function. ICA-1s in plasma serum was measured over the course of 24 h for both oral and intravenous treatments. Heart, liver, kidney, and brain tissues were analyzed for accumulation of ICA-1s. Finally, athymic nude mice were xenografted with DU-145 prostate cancer cells. After tumors reached ~0.2 cm, they were either treated with ICA-1s or left as control and measured for 30 days or until the tumor reached 2 cm. Results showed tumors in treated mice grew at almost half the rate as untreated tumors, showing a significant reduction in growth. In conclusion, ICA-1s is stable, shows low toxicity, and is a potential therapeutic for prostate carcinoma tumors.

Inhibition of atypical protein kinase C­Î¹ 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 PKC­Î¹ was overexpressed in PC samples collected from cancer patients but not in non-invasive prostate tissues, indicating PKC­Î¹ as a possible prognostic biomarker for the progression of prostate carcinogenesis. Immunohistochemical staining further confirmed the association between PKC­Î¹ and the prostate malignancy. The DU­145 and PC­3 PC cell lines, and the non-neoplastic RWPE­1 prostatic epithelial cell line were cultured and treated with aPKC inhibitors 2­acetyl­1,3-cyclopentanedione (ACPD) and 5-amino­1-(1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)­1H-imidazole-4-carboxamide (ICA­1). Western blot data demonstrated that ICA­1 was an effective and specific inhibitor of PKC­Î¹ and that ACPD inhibited PKC­Î¹ and PKC­Î¶. Furthermore, the two inhibitors significantly decreased malignant cell proliferation and induced apoptosis. The inhibitors showed no significant cytotoxicity towards the RWPE­1 cells, but exhibited cytostatic effects on the DU­145 and PC­3 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 PKC­Î¹ and PKC­Î¶ are essential for the progression of PC, and that ACPD and ICA­1 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.

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's 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 TGFß/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.

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, ß-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.