Atypical Protein Kinase Cs in Melanoma Progression

*Wishrawana S. Ratnayake, Christopher A. Apostolatos and Mildred Acevedo-Duncan*

## **Abstract**

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 PKCzeta(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.

**Keywords:** PKC-iota (ι), PKC-zeta (ζ), metastasis, FOXO1, c-Jun

## **1. Introduction**

The protein kinase C (PKC) is a family of Ser/Thr kinases which are involved in transmembrane signal transduction pathways triggered by various extra and intracellular stimuli [1]. Over time, more information has become available since the 1st discovery of PKCs in 1970s. Activation of PKCs may depend on calcium ions and cofactors like the lipid metabolite diacylglycerol (DAG) and phosphatidylserine (PS) [2, 3]. The PKC family consists of fifteen isozymes which are grouped into three on the basis of their co-factor requirements [4, 5]. First group is the conventional PKCs (cPKC) which includes the isoforms alpha (α), beta I (βI), beta II (βII) and gamma (γ) and they require calcium ions, DAG and phospholipids for the activation. Second group is the novel PKCs (nPKC) and it includes delta (δ), epsilon (ε), eta (η) and theta (θ). These are calcium ion independent but dependent on DAG and phospholipids. The aPKC isozymes are the third group, which are independent of Calcium and DAG for their activation. PKC-ζ and PKC-ι in humans

(lambda (λ) is the mouse homologs of iota) are the three aPKCs. Protein kinase D, mu (μ) and some PKC-related kinases (PRK1, PRK2 and PRK3), known as PKN are also considered as PKCs [6].

PKCs have extremely conserved carboxyl-terminal catalytic domain (kinase domain) and PKC isozymes differ from each other on the basis of their aminoterminal (N-terminal) regulatory domain. The N-terminal domain is very important for secondary messenger binding, recruiting the enzyme to the membrane and protein-protein interactions [2]. The pseudosubstrate (PS) domain is located at the N-terminal. PS has a peptide-sequence similar to that of a substrate but lacks alanine in the phosphoacceptor position. In the inactive form of PKCs, the PS prevents complete activation of PKC by blocking the substrate binding pocket [7]. The PS is released upon activation [8, 9]. The activation of PKCs typically involves a cascade of three coordinated phosphorylation events [10, 11]. First, phosphorylation takes place at the activation loop triggered by phosphoinositide-dependent kinase-1 (PDK-1) [12–14]. This initiates a chain reaction that involves autophosphorylation at the turn motif that further stimulates the autophosphorylation at hydrophobic motif of N-terminal [13]. The autophosphorylation at hydrophobic motif is the third and concluding step of the activation.

Atypical PKCs contains two structurally and functionally distinct isozymes in human, PKC-ι and PKC-ζ. The amino acid sequences in both PKC-ι and PKC-ζ are very similar to each other [15, 16]. PKC-ι is encoded by the PRKCI gene and PKC-ζ is encoded by the PRKCZ gene. They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration of carcinoma cells. Additionally, aPKCs play important roles in insulin-stimulated glucose transport [16, 17]. PKC-ι specifically has a strong influence on cell cycle progression. It is also involved in changing cell polarity during cell division [17]. Lung cancer cell proliferation is highly dependent on the PKC-ι level since it increases tumor cell proliferation by activating the ERK1 pathway [15]. PKC-ι and PKC-ζ are expressed in both transformed and malignant melanoma [18]. Overexpression of PKC-ι plays an important role in the chemoresistance of leukemia [19]. PKC-ι is involved in glioma cell proliferation by regulating by phosphorylation of cyclin-dependent kinase activating kinase/cyclin-dependent kinase 7 pathway [20, 21]. A very important study by Selzer et al., investigated the presence of 11 PKC isoforms in 8 different melanoma metastases, 3 normal melanocyte cell lines and 3 spontaneously transformed melanocytes along with several melanoma tumor samples. PKC-ζ and PKC-ι

#### **Figure 1.**

*aPKC expression comparison of normal melanocytes and melanoma cell lines. The expression of PKC-ι and PKC-ζ was reported at approximately 50 and 100% confluency for PCS-200-013 and MEL-F-NEO normal melanocytes against SK-MEL-2 and MeWo metastatic melanoma cells. Western blots were conducted with 50 μg of total proteins loaded in each lane and the complete procedure was adapted from Ratnayake et al. [67].*

**25**

*Atypical Protein Kinase Cs in Melanoma Progression DOI: http://dx.doi.org/10.5772/intechopen.83410*

**cells via NF-κB and PI3K/AKT pathways**

normal melanocytes do not depend on aPKCs.

groups can be found in the latter portion of the chapter.

were found in all transformed melanocytes and melanoma metastases samples in very high levels. PKC-ζ was also found in normal melanocytes in low levels. **Figure 1** demonstrates a comparison of the aPKC expression in two normal melanocyte cell lines (PCS-200-013 and MEL-F-NEO) against two melanoma cell lines (SK-MEL-2 and MeWo) which were used for our studies in Acevedo-Duncan's laboratory at the University of South Florida. As demonstrated in **Figure 1**, normal melanocytes did not show detectable levels of PKC-ι compared to the larger expression observed in SK-MEL-2 and MeWo cell lines. Moreover, PKC-ζ expression was very low in both normal melanocyte cell lines compared to heightened expression in melanoma cells. These results supported the expression patterns demonstrated by patient samples as described in Selzer et al. [18]. All these results indicate a strong relationship between aPKCs and melanoma progression. Here, we discuss our key findings of our recent research on melanoma owing to its relationship with aPKCs in a detailed manner.

**2. Atypical PKCs promote cell differentiation, survival of melanoma** 

Nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and phosphatidylinositol 3-kinase and protein kinase B (PI3K/AKT) pathways are often hyper-activated in many different cancers in order to promote cellular differentiation, growth and survival. Overexpression of aPKCs is often associated with anti-apoptotic effects in many cancers. We have published outcomes of *in-vitro* treatments of aPKC specific inhibitors in which, treatments decreased melanoma cell population markedly compared to normal melanocytes [22–25]. These results confirm that melanoma cellular functions are highly dependent on aPKCs, but

Our recent publications describe the *in-vitro* effects of five aPKC inhibitors on

All five inhibitors were cytostatic to malignant cells rather than cytotoxic. Cells underwent growth arrest before apoptotic stimulation. Regardless, ICA-1S and ICA-1T showed a minor toxicity towards malignant melanoma cells, suggesting

melanoma cell lines compared to normal melanocytes [22, 23]. 2-Acetyl-1, 3-cyclopentanedione (ACPD) and 3,4-diaminonaphthalene-2,7-disulfonic acid (DNDA) are specific to both PKC-ι and PKC-ζ while [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 PKC-ι and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) is specific to PKC-ζ. These compounds were identified from the National Cancer Institute/ Developmental Therapeutics Program (NCI/DTP) database using molecular docking simulations. "AutoDockTools" and "AutoDock Vina" programs were used for the docking simulation by selecting structural pockets in PKC-ι and PKC-ζ which were compatible with small drug like molecules. Sixteen different pockets were identified on PKC-ι and PKC-ζ structures using "fpocket," a very fast open source protein pocket (cavity) detection system based on Voronoi Tessellation. We confirmed the presence of a potentially druggable allosteric site in the structure of PKC-ι using solved crystal structure of PKC-ι. The pocket located in C-lobe of the kinase domain, is framed by solvent exposed residues of helices ⍺F-⍺I and the activation segment. PKC-ι inhibitors were predicted to interact with this site with moderate affinity based on molecular docking. Combinations of drugs targeting the ATP binding site and allosteric sites would be expected to more effectively inhibit cancer cell growth [23]. More details about other aPKC inhibitors form different research

*Atypical Protein Kinase Cs in Melanoma Progression DOI: http://dx.doi.org/10.5772/intechopen.83410*

*Cutaneous Melanoma*

also considered as PKCs [6].

third and concluding step of the activation.

(lambda (λ) is the mouse homologs of iota) are the three aPKCs. Protein kinase D, mu (μ) and some PKC-related kinases (PRK1, PRK2 and PRK3), known as PKN are

PKCs have extremely conserved carboxyl-terminal catalytic domain (kinase domain) and PKC isozymes differ from each other on the basis of their aminoterminal (N-terminal) regulatory domain. The N-terminal domain is very important for secondary messenger binding, recruiting the enzyme to the membrane and protein-protein interactions [2]. The pseudosubstrate (PS) domain is located at the N-terminal. PS has a peptide-sequence similar to that of a substrate but lacks alanine in the phosphoacceptor position. In the inactive form of PKCs, the PS prevents complete activation of PKC by blocking the substrate binding pocket [7]. The PS is released upon activation [8, 9]. The activation of PKCs typically involves a cascade of three coordinated phosphorylation events [10, 11]. First, phosphorylation takes place at the activation loop triggered by phosphoinositide-dependent kinase-1 (PDK-1) [12–14]. This initiates a chain reaction that involves autophosphorylation at the turn motif that further stimulates the autophosphorylation at hydrophobic motif of N-terminal [13]. The autophosphorylation at hydrophobic motif is the

Atypical PKCs contains two structurally and functionally distinct isozymes in human, PKC-ι and PKC-ζ. The amino acid sequences in both PKC-ι and PKC-ζ are very similar to each other [15, 16]. PKC-ι is encoded by the PRKCI gene and PKC-ζ is encoded by the PRKCZ gene. They are believed to be involved in cell cycle progression, tumorigenesis, cell survival and cell migration of carcinoma cells. Additionally, aPKCs play important roles in insulin-stimulated glucose transport [16, 17]. PKC-ι specifically has a strong influence on cell cycle progression. It is also involved in changing cell polarity during cell division [17]. Lung cancer cell proliferation is highly dependent on the PKC-ι level since it increases tumor cell proliferation by activating the ERK1 pathway [15]. PKC-ι and PKC-ζ are expressed in both transformed and malignant melanoma [18]. Overexpression of PKC-ι plays an important role in the chemoresistance of leukemia [19]. PKC-ι is involved in glioma cell proliferation by regulating by phosphorylation of cyclin-dependent kinase activating kinase/cyclin-dependent kinase 7 pathway [20, 21]. A very important study by Selzer et al., investigated the presence of 11 PKC isoforms in 8 different melanoma metastases, 3 normal melanocyte cell lines and 3 spontaneously transformed melanocytes along with several melanoma tumor samples. PKC-ζ and PKC-ι

*aPKC expression comparison of normal melanocytes and melanoma cell lines. The expression of PKC-ι and PKC-ζ was reported at approximately 50 and 100% confluency for PCS-200-013 and MEL-F-NEO normal melanocytes against SK-MEL-2 and MeWo metastatic melanoma cells. Western blots were conducted with 50 μg of total proteins loaded in each lane and the complete procedure was adapted from Ratnayake et al. [67].*

**24**

**Figure 1.**

were found in all transformed melanocytes and melanoma metastases samples in very high levels. PKC-ζ was also found in normal melanocytes in low levels. **Figure 1** demonstrates a comparison of the aPKC expression in two normal melanocyte cell lines (PCS-200-013 and MEL-F-NEO) against two melanoma cell lines (SK-MEL-2 and MeWo) which were used for our studies in Acevedo-Duncan's laboratory at the University of South Florida. As demonstrated in **Figure 1**, normal melanocytes did not show detectable levels of PKC-ι compared to the larger expression observed in SK-MEL-2 and MeWo cell lines. Moreover, PKC-ζ expression was very low in both normal melanocyte cell lines compared to heightened expression in melanoma cells. These results supported the expression patterns demonstrated by patient samples as described in Selzer et al. [18]. All these results indicate a strong relationship between aPKCs and melanoma progression. Here, we discuss our key findings of our recent research on melanoma owing to its relationship with aPKCs in a detailed manner.
