Neil G. Anderson

The protein kinase B/Akt signalling pathway in human malignancy

Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which in mammals comprises three highly homologous members known as PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). PKB/Akt is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. The activation mechanism remains to be fully characterised but occurs downstream of phosphoinositide 3-kinase (PI-3K). PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a lipid second messenger essential for the translocation of PKB/Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. PKB/Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer. Constitutive PKB/Akt activation can occur due to amplification of PKB/Akt genes or as a result of mutations in components of the signalling pathway that activates PKB/Akt. Although the mechanisms have not yet been fully characterised, constitutive PKB/Akt signalling is believed to promote proliferation and increased cell survival and thereby contributing to cancer progression. This review surveys recent developments in understanding the mechanisms and consequences of PKB/Akt activation in human malignancy.

ZD1839 (Iressa), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression

Overexpression of the growth factor receptors EGFR and erbB2 occurs frequently in several human cancers and is associated with aggressive tumour behaviour and poor patient prognosis. We have investigated the effects of ZD1839 (Iressa), a novel EGFR tyrosine kinase inhibitor, on the growth, in vitro and in vivo, of human cancer cell lines expressing various levels of EGFR and erbB2. Proliferation of EGFR‐overexpressing A431 and MDA‐MB‐231 cells in vitro was potently inhibited (50%–70%) by ZD1839 with half‐maximally effective doses in the low nanomolar range. In parallel, ZD1839 blocked autophosphorylation of EGFR and prevented activation of PLC‐γ1, ERK MAP kinases and PKB/Akt by EGF. It also inhibited proliferation in EGFR+ cancer cell lines overexpressing erbB2 (SKBr3, SKOV3, BT474) by between 20% and 80%, effects which correlated with inhibition of EGF‐dependent erbB2 phosphorylation and activation of ERK MAP kinase and PKB/Akt in SKOV3 cells. Oral administration of ZD1839 inhibited the growth of MDA‐MB‐231 and SKOV3 tumours, established as xenografts in athymic mice, by 71% and 32%, respectively. Growth inhibition coincided with reduced proliferation but no change in apoptotic index. Collectively, these results show that ZD1839, at the doses studied, is a potent inhibitor of proliferation not only in cells overexpressing EGFR but also in EGFR+ cells that overexpress erbB2.

The Mitogenic Action of Insulin-like Growth Factor I in Normal Human Mammary Epithelial Cells Requires the Epidermal Growth Factor Receptor Tyrosine Kinase

The signals used by insulin-like growth factor I (IGF-I) to stimulate proliferation in human mammary epithelial cells have been investigated. IGF-I caused the activation of both ERKs and Akt. Activation of ERKs was slower and more transient than that of Akt. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, prevented activation of ERKs but not Akt by IGF-I. Inhibition of the EGFR with function-blocking monoclonal antibodies also specifically blocked IGF-I-induced ERK activation. These effects occurred in primary mammary epithelial cells and in two cell lines derived from normal mammary epithelium but not in mammary fibroblasts or IGF-I-responsive breast carcinoma cell lines. Although IGF-I stimulated the proliferation of both normal and carcinoma cell lines, ZD1839 blocked this only in the normal line. ZD1839 had no effect on IGF-I receptor (IGF-IR) autophosphorylation in intact cells. IGF-I-induced ERK activation was insensitive to a broad spectrum matrix-metalloproteinase inhibitor and to CRM-197, an inhibitor of the EGFR ligand heparin-bound epidermal growth factor. EGFR was detectable within IGF-IR immunoprecipitates from normal mammary epithelial cells. Treatment of cells with IGF-I led to an increase in the amount of tyrosine-phosphorylated EGFR within these complexes. ZD1839 had no effect on complex formation but completely abolished their associated EGFR tyrosine phosphorylation. These findings indicate that IGF-I utilizes a novel EGFR-dependent signaling pathway involving the formation of a complex between the IGF-IR and the EGFR to activate the ERK pathway and to stimulate proliferation in normal human mammary epithelial cells. This form of regulation may be lost during malignant progression.

Growth Hormone-dependent Differentiation of 3T3-F442A Preadipocytes Requires Janus Kinase/Signal Transducer and Activator of Transcription but Not Mitogen-activated Protein Kinase or p70 S6 Kinase Signaling

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42MAPK/p44MAPKmitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70s6k was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70s6k, had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42MAPK/p44MAPKand p70s6k is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

The parathyroid hormone-related protein receptor is expressed in breast cancer bone metastases and promotes autocrine proliferation in breast carcinoma cells

Overproduction of parathyroid hormone-related protein (PTHRP) occurs in a high proportion of primary breast cancers (PBC) and is strongly implicated in their metastatic spread to bone. Although the PTHRP-receptor (PTHRP-R) is often coexpressed with PTHRP in PBC, its role in regulating breast cancer cell proliferation and metastases to bone remains unclear. The aims of this study were to determine the expression of the PTHRP-R in breast cancer bone metastases (BM) and to investigate the effects of PTHRP-R overexpression on breast cancer cell proliferation. PTHRP-R expression occurred in 85% (11 out of 13) of BM compared with 58% (39 out of 67) of PBC. Median expression was higher (P<0.05) in BM compared with PBC. PTHRP increased cAMP accumulation and DNA synthesis in MCF-7 cells stably overexpressing the PTHRP-R (MCF-7WTR) but not in MCF-7VEC control cells. The increase in DNA synthesis was mimicked by the cAMP pathway activator forskolin. The receptor antagonist PTHRP7–34 reduced DNA synthesis in MCF-7WTR cells, but not MCF-7VEC cells, indicating that receptor overexpression promotes autocrine PTHRP activity. MCF-7WTR cells showed increased mitogenic responsiveness to fetal calf serum and reduced doubling times. PTHRP induced weak activation of ERK1 and ERK2 and potentiated their activation by serum growth factors. Collectively these results show that the PTHRP-R is frequently expressed in breast cancer BM and indicate that receptor overexpression drives proliferation via autocrine signals that are mediated via cAMP and ERK pathways.

Wortmannin-sensitive Activation of p70 by Endogenous and Heterologously Expressed G-coupled Receptors

In order to study the regulation of the ribosomal protein S6 kinase, p70, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the α adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human α receptor, with the α agonist UK 14304 led to a transient increase in p70 activity. UK 14304 also activated p70 in a clone expressing the porcine α receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70 in α receptor-transfected and in nontransfected cells. Activation of p70 by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70 by both agents. Activation of p70 by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70 by UK 14304, LPA, and PDGF. These data indicate that p70 is regulatable by G-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70 by such agents appears to involve an isoform of PI 3-kinase.

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes:: Possible involvement of the transcription factor CREB

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

Changes in the expression of guanine nucleotide‐binding proteins during differentiation of 3T3‐F442A cells in a hormonally defined medium

Using specific antisera, the expression of the G protein α subunits, G8, gi1, Gi2, Gi3, and G0, were determined in 3T3‐F442A cells during their differentiation to adipocytes in a hormonally defined medium. Differentiation caused distinct increases in the expression of two Gs isoforms and decreases in the expression of both Gi2 and Gi3. Differentiation also resulted in a 2‐ to 4‐fold increase in forskolin‐stimulated adenylyl cyclase activity and a 15‐fold increase in the response of cells to a β‐adrenergic agonist. The increase in Gs expression was also observed, to a lesser degree, in cells maintained at confluence under conditions where morphological conversion was negligible and the decreased expression of Gi2 and Gi3 and the increased β‐adrenergic responsiveness did not occur.

Agonist-Mediated Tyrosine Phosphorylation of Isoforms of the Shc Adapter Protein by the δ Opioid Receptor

Maximally effective concentrations of the opioid agonist D-ala2-D-leu5-enkephalin resulted in some 2-3-fold enhancement of tyrosine phosphorylation of the p52 Shc adapter protein in a clone of Rat-1 fibroblasts transfected to express stably the murine delta opioid receptor. More limited modifications of the tyrosine phosphorylation status of the p46 and p66 forms of Shc were observed in parallel. Epidermal growth factor caused some 10-12-fold enhancement of tyrosine phosphorylation of p52 Shc and marked increases in the p46 and p66 forms. The effect of D-ala2-D-leu5-enkephalin was prevented by pretreatment of the cells with pertussis toxin and was not observed in non-transfected parental fibroblasts whereas the effect of epidermal growth factor was still manifest in both these situations. Half-maximal effects of D-ala2-D-leu5-enkephalin on p52 Shc tyrosine phosphorylation were produced with sub-nanomolar concentrations, in agreement with previous results on the tyrosine phosphorylation of p44MAPK (Burt et al., 1996). p52 Shc became tyrosine phosphorylated more rapidly than p44MAPK in response to D-ala2-D-leu5-enkephalin and its enhanced tyrosine phosphorylation was maintained for at least 10 min.

PTHrP on MCF-7 breast cancer cells: a growth factor or an antimitogenic peptide?

Sir, We read with interest the publication by Hoey et al (2003), on MCF-7 cell proliferation promoted by the overexpressed PTH/PTHrP receptor, since a study on the effects of PTHrP on MCF-7 cell proliferation is currently in progress in our Lab. We knew that the old and new literature reports contradictory findings in this cancer cell line as far as the proliferative response is concerned; it is surprising, however, that the question is still open in 2003. To our knowledge, the first study on PTHrP and MCF-7 cells is the one by Birch et al (1995), cited in Hoeys paper. However, discordant results had already been presented in 1981 (Cho-Chung et al, 1981). Birchs group found a mitogenic effect of PTH and PTHrP on MCF-7 cells, with a parallel increase in cAMP intracellular levels. Therefore, the work by Hoey et al (2003) appears to support Birchs findings. Conversely, more recent studies, also cited in the paper, show that the cAMP pathway inhibits proliferation in MCF-7 cells (Chen et al, 1998). The recent report by Tovar Sepulveda et al (2002), focused on PTHrP and MCF-7, is particularly instructive here, although it does not meet the question of the different response to PTHrP by the same cell. They clearly demonstrated that PTHrP possesses a double signalling on MCF-7: an antimitogenic pathway mediated by the membrane receptor and a second wave of signalling, pro-proliferative and antiapoptotic, based on nuclear localisation of the peptide. The same dual effect had also been described in vascular smooth muscle cells in 1997 (Massfelder et al, 1997). We believe that this controversial aspect is not discussed enough in Hoeys article. However, we can realise that in May 2002, the date of the first submission, the author could not have read the paper by Tovar Sepulveda et al (2002). On the contrary, Hoey knew and cited the work by Falzon and Du (2000). He suspects that Falzons results about the intracrine effect of PTHrP in PTHrP-transfected MCF-7 cells is of poor physiological relevance, since it is unlikely that the same phenomenon takes place in parental cells as well. We agree that transfection, in general, may confer cell characteristics completely different from those of the parental one and this is the main risk in transfection experiments. However, the same remark could be made to Hoeys results, too. Our preliminary results, summarised in Figure 1, strongly differ from Hoeys ones. In fact, our MCF-7 cells respond to 640 nM PTHrP, in contrast to Hoeys cells, which are unresponsive to 125 nM PTHrP. Figure 1 Proliferative response of MCF-7 breast cancer cells to 640 nM PTHrp 1–40. Cells were grown in MEM in the presence of 2.5% FCS. Chemical inhibitors and activators were used at the following doses: SQ 22536: 50 μ ... This is the first difference with Hoeys work. On working with parental MCF-7 cells, we needed to increase the PTHrP dose to obtain some effect on proliferation. We also failed to appreciate significant variations below the dose of 640 nM. Thus, MCF-7 cells are not unresponsive to PTHrP; they simply need a stronger treatment. Our results (still incomplete to be collected in a manuscript) also indicate that exogenous PTHrP triggers two main transduction pathways: the adenylyl-cyclase (AC)/protein kinase (PK) A and the phospholipase (PL) C/PKC cascade. Thus, the native PTH/PTHrP receptor is coupled also to the Ca2+ signalling route in MCF-7 cells. This is the second difference: Hoeys cells have probably lost this transduction pathway. In our MCF-7 cells, both PKA and PKC are antimitogenic and this appears to be the main difference. We cannot tell, at the moment, which is the major player, but the selective block of AC or PLC reduces the antiproliferative effect of PTHrP. We chose to block the membrane enzymes, rather than PKs, because, as reported also by Hoey, chemical inhibitors of PKs are toxic for cells and mask the proliferative effect of a test factor. This is the only experience we have in common with Hoeys group. Hoey describes an effect of PTHrP on cell sensitivity to growth factors, but this is not a novel phenomenon, as it was already described by Linseman et al (1995). Probably, Hoey is dealing with the so-called transactivation of growth factors tyrosine kinase (TK) receptors by PTHrP G-protein-coupled receptor (GPCR) (Linseman et al, 1995; Lowes et al, 2002). We observed a similar phenomenon in 2002 while culturing skin fibroblasts in the presence of serum (then of growth factors) (Maioli et al, 2002). We do not have the presumption to solve the conflict between Hoeys and our results, but in an attempt to reconcile them, we hypothesise that the overexpressed receptor, unable to couple to PLC, thus lacking one of the two antiproliferative pathways, acquired enhanced ability to transactivate TK receptors; the pro-mitogenic effect predominates in the presence of 2% serum. We believe that such a pro-proliferative action of PTHrP, mediated by the membrane receptor, is operative also in parental MCF-7 cells. In fact, in our preliminary study, exogenous 1–40 PTHrP shares pro-mitogenic properties in 2.5% serum-cultured MCF-7 cells, when the membrane AC and PLC are simultaneously blocked. Finally, the main question and the only one that cannot be ascribed to the transfection process is the opposite effect of forskolin (an AC activator) on parental (ours) and transfected MCF-7 cells (Hoeys ones). In fact, it is now clear that cAMP can exert both pro- and antimitogenic effects acting at the level of different Raf isoforms. As a rule, the cAMP/PKA pathway stimulates Ras-independent and Rap-1-dependent extracellular regulated kinase (ERK) phosphorylation and cell proliferation in Raf-B-expressing cells, but it inhibits growth in Raf-B-negative cells (Fujita et al, 2002). Then, is the difference in cAMP levels sufficient to explain the discrepancy? Our previous experience on skin fibroblasts, which express predominantly the Raf-1 isoform (Fortino et al, 2002), indicates that forskolin (then cAMP) is always antiproliferative, at any dose tested (Maioli et al, 2002). Consistently, in our MCF-7 cells, forskolin (as well as phorbol 12-myristyl 13-acetate, PMA) mimics the PTHrP antimitogenic effect. Although a study focused on Raf isoforms in MCF-7 cells is still missing, Raf-1 isoform is surely expressed (Weinstein-Oppenheimer et al, 2001). Clarifying this and the other aspects would add significantly to our present biological and clinical knowledge on PTHrP role and responsibility in cancer.