Wishrawana S. Ratnayake

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 862: Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma

Melanoma is a type of cancer occurs in melanocytes. Approximately 90% of melanoma occurs in skin (cutaneous melanoma) but can rarely arise from the mucosal surfaces or areas which neural cells migrate. Examples are eye, intestine and mouth [Eur. J. Cancer, 69: 39-42 (2016)]. 76,380 of new cases and 10,130 number of deaths are expected in 2016 in the USA due to melanoma [http://seer.cancer.gov/statfacts/html/melan.html (11/05/2016)]. Atypical PKCs contains two structurally and functionally distinct isozymes in human which are PKC-ι (iota) and PKC-ζ (zeta). They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration. We believe that atypical PKCs play an important role in cell motility of melanoma by involving the signaling pathways which induces EMT-type III (Epithelial to Mesenchymal Transition). In normal melanocytes, PKC-ζ was found in low levels and PKC-ι was not detected. But both proteins are detected in very high levels in malignant melanoma [Melenoma Res. 12:201-209 (2002)]. In the current study, we have investigated the effects of novel atypical PKC inhibitors [4-(5-amino-4-carbamoylimidazol-1-yl)-2, 3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) which is specific to PKC-ι and 8-hydroxy-1, 3, 6-naphthalenetrisulfonic acid (Compound-50) which is specific to PKC-ζ on the cell proliferation, apoptosis and cell migration of two malignant melanoma cell lines (SK-MEL-2 and MeWo) compared to a normal melanocyte cell line (PCS-200-013). We showed that both inhibitors can decrease the levels of total and phosphorylated levels of PKC-ζ and PKC-ι. Furthermore, both inhibitors increased the levels of E-cadherin and decreased the levels of Vimentin which is a mesenchymal marker associated with EMT. Treatments with inhibitors altered the levels of CD44, a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, migration and tumor cell homing during metastasis. These results suggest that both PKC-ι and PKC-ζ are involved in signaling pathways which upregulate EMT and which can be effectively suppress using ICA-1 and Compound-50. Furthermore we established that treatment with ICA-1/Compound-50 induced apoptosis as shown by increasing Caspase-3 levels and decreasing Bcl-2 levels. Citation Format: Wishrawana Sarathi Ratnayake, Mildred Acevedo-Duncan. Atypical protein kinase c inhibitors can repress epithelial to mesenchymal transition (type III) in malignant melanoma [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 862. doi:10.1158/1538-7445.AM2017-862

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 4569: Use of ACPD and ICA-1 as inhibitors of atypical proteinkinase C-zeta (ζ) and iota (ι) in metastasized melanoma cells

The number of melanoma cases report is increased every year. Malignant melanoma is very common among all Caucasian populations worldwide. New cases in these populations are expected to be doubled every 10-20 years. According to the NIH Surveillance, Epidemiology and End Results (SEER) program, 73,870 new cases and 9,940 deaths were reported in 2015 in USA [http://seer.cancer.gov/statfacts/html/melan.html (11/10/2015)]. It is not totally understood which intracellular chemicals are involved in certain signaling pathways and which governs the metastasis of melanoma cancer cells. Even though PKC- iota (ι) was not reported in normal melanocytes, it was detected in high amounts in both transformed melanocytes and melanoma metastases. PKC- zeta (ζ) was also reported in both normal melanocytes and melanoma metastases [Melanoma Res. 12:201-209 (2002)]. We believe these atypical PKCs play an important role in cell motility of melanoma therefore we tested two inhibitors for them. The objective of this study was to test the inhibition effectiveness of ACPD [Diabetes. 63:2690-2701 (2014)] on both PKC-ι and PKC-ζ and ICA-1 [Int. J. Biochem. Cell Biol.43:784-794(2011)] as an inhibitor of PKC-ι. SK-MEL-2 metastasis melanoma cells and PCS-200-013 normal melanocyte cells were cultivated and treated with ACPD and ICA-1 in separate sets of flasks for three consecutive days while taking the cell count for every 24 hrs. Preliminary results of this experiment shows statistically significant decrease in cell number in SK-MEL-2 cells while no change in PCS-200-013. Future investigations will involve examining the effects of ACPD and ICA-1 on cell motility. Our preliminary results confirms the cell population of melanoma cells have an inversely proportional relationship with the drug concentrations. In conclusion, ACPD and ICA-1 are capable of decreasing the cell proliferation of melanoma cancer cells. Citation Format: Wishrawana S. Ratnayake, Mildred Acevedo-Duncan. Use of ACPD and ICA-1 as inhibitors of atypical proteinkinase C-zeta (ζ) and iota (ι) in metastasized melanoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4569.