Endocrine-Related (2003) 10 389–396 REVIEW 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 C (PKC) is a familyof /threonine that is involved in the transduction of signals for 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 and reviews the preclinical and clinical data available for the efficacyof PKC inhibition. Endocrine-Related Cancer (2003) 10 389–396

Introduction anchoring (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 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 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: 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 . 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 (NHL), express significantly higher levels of PKC than ER-positive and lung cancer (Fuse et al. 1998). breast cancer cell lines (Fabbro et al. 1986, Platet et al. 1998). In two cell lines PKCδ appears to play a fundamental PKC412 role in the regulation of growth in ER-positive breast cancer cells (Shanmugam et al. 2001). Furthermore, preliminary It seems likely that anticancer drugs acting as signal trans- data suggest that PKCα overexpression may predict resist- duction inhibitors rather than as classic cytotoxics may ance to (Tonetti et al. 2002). These studies suggest require prolonged exposure to be effective. In this context that members of the PKC family have potential as targets in PKC412 has the advantage of being administered orally. the treatment of breast cancer. PKC412 was well tolerated in the phase I study, the main toxicities being nausea, vomiting, fatigue and diarrhoea. A PKC inhibitors formal maximum-tolerated dose (MTD) was not defined (Propper et al. 2001). Like UCN01, PKC412 has a longer The PKC family is undoubtedly an attractive target for thera- half-life than would be predicted from preclinical studies due peutic intervention given its role in tumorigenesis and the to altered gastrointestinal absorption and plasma protein potential for enhancing cytotoxicity of existing drugs. The binding (in particular binding to AAG) in patients with existence of different isozymes provides an opportunity to cancer (Propper et al. 2001). Given the lack of classic cyto- develop pharmacological agents that target specific PKC toxic side-effects and unusual pharmacokinetics, assessing functions. Equally, however, there is no doubt that the com- the inhibitory effects of PKC412 on PKC signalling path- plex nature of the many secondary messenger systems that ways in vivo in parallel with standard pharmacokinetic involve PKC renders selective drug action difficult. Few of studies was crucially important. The release of tumour the currently available pharmacological agents exhibit a high factor (TNF) and interleukin (IL)-6 from phyto- degree of selectivity for a specific PKC isoform, and the haemagglutinin-stimulated whole blood cells were both sig- majority also act on other PKs. The principal agents under nificantly inhibited in a time- and dose-dependent manner investigation are listed in Table 1. with the level of inhibition appearing to plateau at doses over The microbial alkaloid staurosporine was identified 20 100 mg/day. In addition, down-regulation of extracellular years ago as an antiproliferative agent and potent inhibitor signal-related kinase 2 (ERK2) was demonstrated across the of PKC. It probably acts by competing at PKC’s conserved 50–300 mg/day dose levels (Thavasu et al. 1999). The inves- ATP-binding sites. Although its specificity for PKC and its tigators concluded that, although a formal MTD had not been isoforms is poor, staurosporine has served as a lead com- identified, the increased prevalence of symptomatic toxicities www.endocrinology.org Downloaded from Bioscientifica.com at 09/24/2021391 07:51:41PM via free access Mackay and Twelves: Protein kinase C

Table 1 Protein kinase C inhibitors Drug Class Specificity/selectivity Comments References Staurosporine Poor, inhibitorytowards Potent, poor Way et al. (2000) serine/threonine and selectivity tyrosine protein kinases PKC412 PKCs α, β, γ, δ, ε, η. Potentiates, Takahashi et al. (1989), Also inhibits tyrosine , Andrejauskas & Regenass kinase pathways (1992), Mizuno et al. (1993), Utz et al. (1994), Budworth et al. (1996) UCN01 cPKCs>nPKCs Potentiates Mizuno et al. (1993), platinum, mitomycin Bunch & Eastman (1996), C, camptithecin, Hseuh et al. (1998), 5-FU Jones et al. (2000), Sugiyama et al. (2000) Go6976 cPKCs>nPKCs — Martiny-Baron et al. (1993) Bryostatin Macrocyclic lactone Activator of cPKC and Potentiates cytosine Hornung et al. (1991), nPKCs; in presence of arabinoside, Elgie et al. (1998), activating ligands acts , McGowan et al. (1998), as an antagonist tamoxifen, Wang et al. (1998), Mohammed et al. (2000) Tamoxifen Non-steroidal PKCs α, β, γ — Bignon et al. (1989), anti-oestrogen non-selective O’Brian et al. (1985) Bisindolylmaleimide Indolocarbazole PKCβ Used in treatment Wilkinson et al. (1993) LY333531 of Salfingol Synthetic lipid — Preclinical: little Schwartz et al. (1995), tumour effect Kedderis et al. (1995) administered alone Potentiates doxorubicin Antisense 19 mer Dean et al. (1994, 1996) oligonucleotides phosphorothioate oligodeoxynucleotide ISIS3521 PKCα Mckay et al. (1999) ISIS9606 PKCα Mckay et al. (1999)

at 225 mg/day along with the apparent plateau in drug expo- ficulties inherent in selecting pharmacodynamic endpoints in sure and biological activity were such that 150 mg/day clinical trials. should be the recommended phase II dose (Propper et al. 2001). This dose was evaluated in a phase II trial of PKC412 Bryostatin in patients with accessible melanoma which incorporated tumour biopsies. This study failed to demonstrate consistent The bryostatins are a family of at least 20 naturally occurring target inhibition or pharmacodynamic efficacy. Tumour cyto- macrocyclic lactones derived from the marine bryozoan solic PKC inhibition varied from 7 to 91% in seven out of Bugula neritina. They have been shown to have promising nine patients examined and tumour particulate PKC activity antineoplastic and immunomodulatory activity in preclinical was reduced by 11–79% in four patients. In vitro testing models. Bryostatin I is the prototype of this novel class of demonstrated less than 10% inhibition of multi-drug resist- agents (Schaufelberger et al. 1991). They are potent acti- ance (MDR) (Propper et al. 2001). Although the phase I vators of cPKC and nPKC subfamilies. However, in the pres- study results had been encouraging, these negative findings ence of activating ligands, such as the tumour-promoting cast doubt on whether PKC412 achieves its intended effect phorbol esters, bryostatins act as antagonists (Smith et al. in tumours. The development of PK412 illustrates the dif- 1985). The basis for the divergent activities of bryostatin I is

392 Downloaded from Bioscientifica.comwww.endocrinology.org at 09/24/2021 07:51:41PM via free access Endocrine-Related Cancer (2003) 10 389–396 not known but may derive from differential isoform acti- There was no relationship between dose, maximum concen- vation (Szallasi et al. 1994) or nuclear translocation tration of drug, or area under the curve and coagulation times (Hocevar & Fields 1991). Furthermore, bryostatin I potently or complement levels. Dose escalation was discontinued downregulates cPKC/nPKC activity, probably via ubiquitin- because of the attainment of peak plasma concentrations directed proteasomal degradation of the (Lee et al. which approached that associated with complement acti- 1996). Bryostatin may also activate effector cells of the vation in primates. No dose-limiting toxicities were identified immune system and stimulate cytokine production (Mohr et and two patients with NHL achieved complete responses al. 1987, Trenn et al. 1988). (Neumunaitis et al. 1999). A small phase II trial of ISIS3521 Bryostatin 1 has been widely investigated in a series of (2 mg/kg per day administered as a continuous infusion for phase I trials with infusion times varying from 1 to 24 h 21 out of 28 days) in patients with metastatic breast cancer (Philip et al. 1993, Prendiville et al. 1993, Jayson et al. did not show any activity (Gradishar et al. 2001). 1995). The dose-limiting toxicity in all studies has been myalgia with localised phlebitis a problem using the shorter infusion times. Antitumour activity has been demonstrated in The future for PKC inhibitors patients with melanoma, epithelial ovarian carcinoma and The PKC family undoubtedly represents an interesting and NHL (Philip et al. 1993, Prendiville et al. 1993, Jayson et challenging target for the development of novel therapeutic al. 1995). agents. The current PKC inhibitors that have reached the Significant increases in plasma concentrations of TNF-α clinic are relatively non-specific in their actions, do not fully and IL-6 were observed when bryostatin, 50 µg/m2, was exploit the potential for differential inhibition of PKC func- administered as a 1 h infusion weekly for 3 weeks out of tions or specific isozymes and have encountered PK prob- every 4 (Philip et al. 1993). A further study of a weekly 24 h lems such as AAG binding. In the future it may be possible infusion of bryostatin demonstrated significant changes in to develop agents that target a single isoform, different acti- activated PKC in peripheral mononuclear cells during the vating pathways or specific membrane interactions (specific infusion (Jayson et al. 1995). The previously reported elev- RACKs or RICKs). Furthermore, as the downstream events ation of IL-6 and TNF-α was not confirmed in this study. resulting from PKC activation are better characterised it may However, an increase in IL-2-induced proliferative response be appropriate to target events further down the signalling in peripheral blood lymphocytes and enhanced LAK activity pathways. Rational synthesis of molecules that target specific was demonstrated. sites within the PKC isoforms is in progress. Peptide frag- Bryostatin showed some evidence of clinical activity in ments that act as inhibitors or activators of translocation and phase I trials but this was not confirmed in phase II trials of antisense approaches are also being pursued (Way et al. patients with pretreated metastatic melanoma (Propper et al. 2000). Success with other inhibitors such 1998) and metastatic (Zonder et al. 2001). as Herceptin and STI571 have demonstrated that develop- It had limited efficacy against relapsed low grade NHL ment of the right agent for the right target can be successful (Varterasian et al. 1998). (Baselga 2001, Verweij et al. 2001). The role of PKC in tumorigenesis and apoptosis suggests that combining PKC Tamoxifen inhibitors with conventional cytotoxics may be effective. Evidence from cell line data and the early phases of clinical The anti-oestrogen tamoxifen is a non-selective inhibitor of trials suggests promising results for the combination of con- PKC. High, PKC inhibitory, doses of tamoxifen achieved ventional cytotoxics with the existing PKC inhibitors (Alkan disease stabilisation of childhood malignant gliomas in a et al. 1993, Bunch & Eastman 1996, Wang et al. 1998, Ilson phase I (Couldwell et al. 1996, Pollack et al. et al. 2000). In addition, PKC412 in vitro sensitises trans- 1997). formed murine fibroblasts to radiation-induced apoptosis (Zaug et al. 2001). It is not clear if this is due to PKC inhi- ISIS3521 bition, but if it is the role of PKC isozyme inhibition in radio- sensitisation would warrant further investigation. Antisense oligonucleotides have been developed to achieve Further translational research is needed to establish if PKC isoform-specific inhibition by inhibiting expression of inhibition of PKC isozymes will prove beneficial in altering target mRNA sequences. ISIS3521, an antisense phos- the natural history of malignancy. phorothioate oligonucleotide to PKCα, has been investigated in a phase I trial. Patients received 2 hourly i.v. infusions (0.15–6 mg/kg per day) three times per week. Drug-related References toxicities included nausea, vomiting, fever, chills and fatigue. 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