Endocrine-Related Cancer (2003) 10 389–396 REVIEW Protein kinase C: a target for anticancer drugs? HJ Mackay and CJ Twelves Department of Medical Oncology, Cancer Research UK Beatson Laboratories, Alexander Stone Building, Switchback Road, Glasgow G61 1BD, UK (Requests for offprints should be addressed to C Twelves; Email: [email protected]) (HJ Mackayis now at Department of Drug Development, Medical Oncology,Princess Margaret Hospital, Toronto, Ontario, Canada) (CJ Twelves is now at Tom Connors Cancer Research Centre, Universityof Bradford, Bradford, UK) Abstract Protein kinase C (PKC) is a familyof serine/threonine kinases that is involved in the transduction of signals for cell proliferation and differentiation. The important role of PKC in processes relevant to neoplastic transformation, carcinogenesis and tumour cell invasion renders it a potentiallysuitable target for anticancer therapy. Furthermore, there is accumulating evidence that selective targeting of PKC mayimprove the therapeutic efficacyof established neoplastic agents and sensitise cells to ionising radiation. This article reviews the rationale for targeting PKC, focuses on its role in breast cancer and reviews the preclinical and clinical data available for the efficacyof PKC inhibition. Endocrine-Related Cancer (2003) 10 389–396 Introduction anchoring proteins (Csuki & Mochley-Rosen 1999, Way et al. 2000). The protein kinase C (PKC) family consists of at least 12 The phospholipid diacylglycerol (DAG) plays a central isozymes that have distinct and in some cases opposing roles role in the activation of PKC by causing an increase in the in cell growth and differentiation (Blobe et al. 1994, Ron & affinity of PKCs for cell membranes which is accompanied Kazanietz 1999). Early observations that PKC isozymes are by PKC activation and pseudo-substrate release (Newton activated by tumour-promoting phorbol esters (Castagna et 1995). Activated PKC then phosphorylates and activates a al. 1982) suggested a key role for PKC in tumour promotion range of kinases. Signals that stimulate G-protein-coupled and progression. This led to PKC being considered as a target receptors, receptor tyrosine kinases and non-receptor protein for anticancer therapy. The presence of PKC isoforms with tyrosine kinases can all cause the production of DAG differential activation and tissue distribution (Way et al. (Nishizuka 1992, Newton 1995). Several PKC isotypes are 2000) raised the possibility of developing PKC isozyme- activated independently in a redundant manner through the specific inhibitors with the potential for targeting specific phospholipase C (PLC) and phosphatidylinositol 3-kinase intracellular pathways. (PI3K) pathway (Newton et al. 1995). For example, platelet- PKC was originally identified as a phospholipid- and cal- derived growth factor activates PKCε through redundant and cium-dependent protein kinase (PK) (Takai et al. 1979). The independent signalling pathways involving PLCγ or PI3K subsequent classification of the isoforms is based on struc- (Moriya et al. 1996). There is some evidence to suggest that tural and activational characteristics (Nishizuka 1992, there are functional differences between PKC activated Newton 1995). The PKCs are divided into three subfamilies: through different pathways (Ohno 1997). conventional or classic PKCs (cPKC), non-classic or novel The downstream events following PKC activation are PKCs (nPKC) and atypical PKCs; their binding and acti- little known. The main pathway, which is activated by PKC, vational characteristics are summarised in Fig. 1 (Schenk & is the MEK-ERK pathway. PKCα phosphorylates and acti- Snaar-Jagalska 1999). Activation of PKC isoforms results in vates the serine/threonine kinase Raf1 both in vitro and in changes in their subcellular location. For example, PKCα vivo (Kolch et al. 1993). It is, however, unclear how this and PKCζ translocate from the cytosol to the perinuclear phosphorylation contributes to activation of Raf1. In membrane on activation (Distanik et al. 1994). Cell-specific addition, It has been proposed that PKCα, δ and ε also acti- isoform functions may be conferred by differences in subcel- vate the MEK-ERK pathway via Raf1 (Ueda et al. 1996, lular localisation following translocation to specific Cai et al. 1997). Activated Raf1 phosphorylates MEK1 and Endocrine-Related Cancer (2003) 10 389–396 Online version via http://www.endocrinology.org 1351-0088/03/010–389 2003 Societyfor Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 07:51:41PM via free access Mackay and Twelves: Protein kinase C θ (i) conventional PKC: Ras Rac 1 PKC Regulatory domain Catalytic domain C1V1 V2C2 V3C3 V4 C4 V5 PAK PKCα, βI, γ PKC α,δ,ε Raf MEKK1 N C GSK 3β DAG, phorbol Ca++ ATP Substrate MEK1a SEK1/MKK4 ester binding binding; binding binding pseudo- anchoring ERK1,2 SAPK/JNK (ii) novel PKC: C2-like region in V1 (iii) atypical PKC: C2-like region in V1 only 1 zinc finger in C1 region Cell transcription and cell proliferation R1, R2 = H: Staurosporine R1=OH, R2 =H: UCN01 Bryostatin 1 R1=H, R2 = benzoyl group: PKC412 Figure 1 Schematic representation of PKC structure. MEK2, which activate the mitogen-activated protein kinase breast (O’Brian et al. 1989), lung (Takenaga & Takahashi cascade, ultimately resulting in transcription of genes 1996) and gastric carcinomas (Schwartz et al. 1993). In vivo, involved in cell proliferation (Marshall 1996). PKCα, βI and however, the relationship is less clear, with PKCβ expression γ specifically inactivate GSK-3β by phosphorylation, leading being found to increase, remain the same or decrease in colon to derepression of the cJun transcription factor (Goode et al. tumours when compared with normal epithelium. This may 1992). PKCθ is reported to synergise with the partially be explained by changes occurring in PKC expres- Ca2 +-dependent phosphatase calcineurin to stimulate JNK1 sion during tumorigenesis. In colon cancer, PKCβII appears via Rac1 (Werlen et al. 1998). to be overexpressed early in the carcinogenic process with PKCα and βI expression decreasing later in tumour develop- Role of PKC in cancer ment (Gokmen-Polar et al. 2001). Studies with antisense PKCα oligonucleotides have demonstrated inhibition of Early studies suggested a role for PKC isozymes in tumour tumour growth in nude mice bearing implanted human glio- promotion. PKC isozymes are selectively activated by blastomas and human bladder, lung and colon carcinomas tumour-promoting phorbol esters in a way similar to the (Dean et al. 1996). In vitro experiments suggest that PKCα endogenous activator DAG (Castagna et al. 1982). Subse- inhibition may result in apoptosis. PKCδ has also been impli- quently a direct correlation between the ability of individual cated in the apoptotic response in human prostate cancer and phorbol esters to promote tumours and to activate PKC has myeloid leukaemia cell lines (Fujii et al. 2000, Majumder et been demonstrated (Nishizuka 1984). Furthermore, other al. 2000). Inhibition of PKC may have an impact on the structurally unrelated tumour promoters (such as teleocidin) invasive and metastatic potential of malignant cells. The also activate PKC at high concentrations (Nishizuka 1984). PKC family is involved in cellular adhesion with PKC Increased levels of PKC have been associated with inducing the expression of adhesion proteins (Lane et al. malignant transformation in a number of cell lines including 1989) and many cell adhesion receptors act as PKC sub- 390 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/24/2021 07:51:41PM via free access Endocrine-Related Cancer (2003) 10 389–396 strates (Herbert 1993). Atypical PKCs have been implicated pound from which many novel PKC inhibitors have been in the evolutionarily conserved PAR protein complex and developed (Way et al. 2000). Drugs such as PKC412 play a critical role in the development of junctional structures (N-benzoyl-staurosporine) and UCN01 (7-hydroxystauro- and apico-basal polarisation of mammalian epithelial cells sporine) exhibit improved selectivity, with a potentially (Suzuki et al. 2001). Furthermore, Wnt5a appears to be up- better therapeutic index in vivo and have been reported to regulated in breast cancer by activated PKC and down- enhance the effects of other cytotoxic agents. regulated by its inhibition (Jonsson et al. 1998). Thus PKC may act via the Wnt genes to affect the cytoskeleton and UCN01 cellular adhesion. UCN01 was administered in phase I trials as either a 3 or Role of PKC in breast cancer 72 h i.v. infusion every 21–28 days (Fuse et al. 1998). The recommended phase II dose for UCN01 administered as a In vitro studies suggest a positive correlation between eleva- 72 h infusion is 42.5 mg/m2 per day. UCN01 exhibits unusual ted PKC levels and both the invasive and chemotactic poten- pharmacokinetics in man compared with animal models with tial of human breast cancer cell lines (Blobe et al. 1994). long elimination half-lives of between 253 and 1660 h. The One small study in nine patients examined PKC levels in unexpected results obtained with UCN01 in man can be surgical specimens of human breast tumours compared with explained by high binding to AAG resulting in < 0.02% free normal breast tissue obtained from the same patient. PKC UCN01, compared with 0.49–1.75% in animal studies (Fuse activity was significantly higher in the tumour tissue com- et al. 1998, 1999). Dose-limiting toxicities of pulmonary dys- pared with the normal tissue (P = 0.0003) (O’Brian et al. function, nausea and vomiting, and hypergylcaemia with 1989). PKC may also be associated with hormone depen- metabolic acidosis were reported. Preliminary evidence sug- dence in breast cancer cells. Stable transfection of PKCα ren- gests UCN01 modulation of both PKC substrate phosphory- ders T47D human breast cancer cells hormone-independent lation and the DNA damage-related G2 checkpoint (Sausville in vitro and in vivo (Tonetti et al. 2000). Several oestrogen et al. 2001). Antitumour activity was observed against leio- receptor (ER)-negative human breast cancer cell lines myosarcoma, non-Hodgkin’s lymphoma (NHL), melanoma express significantly higher levels of PKC than ER-positive and lung cancer (Fuse et al.