Metbolic Control in Cancer Regulation in Metabolism Group Colloquium Edited by K. Snell (University of Surrey) and Organized by K. Snell (University of Surrey) and D. H. Williamson (Nuffield Department of Clinical Medicine, Oxford)

Signal transduction mechanisms in cancer HANS GRUNICKE lrisiitirte for Merlicul C'heinisiy uiid Hiochetnistty Uiiiversity of Iritisbruck, A -6020 1iiii.shrirck. Austriu

Evidetice for u rus-tiirdiuted cmstitiitivo trc,tiLwioii o f eleiricvirs

of'growth factor sigiiul truizsdirciioii

The supposition that products of the rus gene family (Harvey, Kirsten, N-rus) may be involved in postreceptor signal transduction is based o n their remarkable homology Malignant growth has in many cases been shown to be due with known GTP-binding proteins (so-called G-proteins) (for to autonomous self-stimulation of cellular proliferation. reviews see [ 12. 131). This homology includes structural Three basic mechanisms have been elucidated each of which (amino acid sequence) as well as functional features (GTP/ is capable of leading to growth autonomy: ( i ) autocrine pro- GDP binding, intrinsic GTPase activity). Since G-proteins duction of growth factors; (ii) synthesis of abnormal growth have been identified as signal transducing elements acting t o factor receptors which, even in the absence of the corre- couple membrane receptors with cellular effector systems. it sponding ligands, behave as their activated normal counter- is possible to postulate a similar role for the product of the parts; and (iii) constitutive activation of elements of growth proto-oncogene p2 1 '"'. Furthermore. it is intriguing t o factor signal transduction cascades. Various oncogenes seem assume that the altcred p2 I encoded by the mutated, transto act by one of these mechanisms (for reviews see [ 11 and formed rus oncogene niight act as a Constitutively activated [2]).This paper focuses on those oncogenes for which evi- G-protein triggering a mitogenic pathway. This hypothesis is dence for a constitutive activation of elements of growth supported by data demonstrating that injection of anti-rus factor signalling mechanisms exists. antibody inhibits the mitogenic activity of serum growth Cells transformed by the avian erythroblastosis virus con- factors [14]. Additional support comes from findings with tain the product of the viral oncogene v-erh-B which reprc- ras-transformed cells providing evidence for an elevated sents a truncated epidermal growth factor receptor [3-5]. turnover of inositide polyphosphates [ 15, 161. Since a variety Although, definitive proof is still lacking, the data available of growth factors are known to act via a phosphatidylinositol bisphosphate-specific phospholipase C, which has also been s o far are consistcnt with the assumption that the v-orb-B product behaves like a constitutively activated epidermal shown to be under control of a G-protein [ 17-2 1 1, these data growth factor-receptor 16, 7 I. An analogous situation is fit well into the original working hypothesis. From the data found in v-fins-transformed cells. The c-fins gene product obtained with rus-transformed cells, however, it is difficult to has been identified as the receptor for colony stimulating decide whether the observed metabolic alterations are direct, factor I , a mycloid cell growth factor [ 81. Oncogene products immediate activities of p2 1"" or whether they represent which may affect growth factor signalling at a postreceptor secondary metabolic alterations associated with a transstage include the src-, uhl-, and fix-gene families 161. The formed phenotype. To address this question, the transformsupposition that these proteins cause a constitutive activation ing oncogene as well as the non-transforming of growth factor signal transduction pathways is based on the proto-oncogene were recombined iiz vitro with the mouse fact that they all represent tyrosine kinases. As several mammary tumour virus long terminal repeat (MMTV-LTR), growth factor receptors have been found to contain tyrosine cloned and used for transfection of NIH 3T3-fibroblasts 1221. kinase activity, it is presumed that tyrosine phosphorylation The MMTV-LTR sequence subjects the oncogene to transis involved in growth control. So far. however, neither thc criptional control by glucocorticoids. Thus the fibroblasts essential substrates of the kinases nor thc physiological transfected with these constructs represent a system in which signals which regulate their activity have been idcntified. expression of the oncogene can be switched o n and off under The mos and raf oncogenes encode serinelthreonine- controlled conditions. specific protein kinases [9, lo]. Protein kinase C is an Expression of the oncogene in transfected NIH 3T3example of a serine kinase which is involved in growth con- fibroblasts by dexamethasone leads to a serum growth trol [ 1 1 ]. Therefore, these proteins, like the tyrosine kinases, factor-independent cellular replication 1231. The cells may equally affect growth factor signalling mechanisms. But acquire a transformed phenotype, grow in soft agar, lose conagain, the precise mechanism by which these proteins inter- tact inhibition and cause tumours after inoculation into athymic mice [ 231. To investigate the biochemical effects fere with growth regulation is obscure. Evidence is accumulating that the products of the rus gene caused by the expression of the rus oncogene, we measured family may indeed be engaged in postreceptor signal trans- the effects o n polyphosphoinositidc metabolism. protein ducing mechanisms, Therefore, this article will focus on kinase C, the Na+/H antiporter and the intracellular Ca'+ p2 1"' and its effects on signal transduction mechanisms. mobilizing system. The studies revealed that expression of the transforming oncogene leads to a growth factor-independent elevation o f Ahhrcviations used: MMTV-LTK. mouse mammary turnour virus phosphatidylinositol bisphosphate turnover, a concomitant long terminal repeat; IP,, inositol I ,4,5-trisphosphate; GAP, G l P a s e activating protein. increase in inositol polyphosphates and a growth factor-inde1iiirodiu.tioii



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pendent stimulation of the N a + / H+-antiporter with a subsequent increase in cytosolic pH 123-251. None of these effects was observed in cells containing the proto-oncogene constructs. There is evidence from our laboratory as well as others (26-281 for an activation of protein kinase C by Harus. A stimulation of protein kinase C has in many cases been shown to activate the Na+/H+-antiporter[29, 301. Therefore, it was quite surprising to observe that the Ha-rusinduced, amiloride-sensitive cytosolic alkalinization is unaffected by inhibitors of protein kinase C or by protein kinase C depletion [31]. It must be concluded that Ha-rus activates the N a + / H+-antiporter by a protein kinase Cindependent pathway which has still to be characterized. The elucidation of this mechanism may reveal Ha-rus-specific early signals which have not been considered s o far. However, the data do not allow the conclusion that the activation of protein kinasc C by Ha-nrs is an irrelevant or dispensable phenomenon. Ha-rus-induced cell replication is depressed by inhibitors of protein kinase C 1261. This is in line with findings demonstrating that, although the formation of the transformed phenotype after expression o f Ha-rus is a protein kinase C-independent phenomcnon. protein kinase C is required for the progression of cells into the S-phase of the cell cycle 132). Expression of v-mos in MMTV-LTR-v-mos transfected fibroblasts has been shown t o cause a similar amiloridesensitive cytosolic alkalinization to that observed after induction of p21"" [23]. Thus, the activation of the Na+/H antiporter is not an Ha-rus-specific phenomenon. Whether v-mos and Ha-rus affect the antiporter via a common mechanism is still unclear. Stimulation by many mitogens has been shown to lead to two important ionic signals: an increase in intracellular pH and a rise in cytosolic C a ? + . This raises the question of whether rus expression also has an effect on cytosolic Ca?+. Inositol 1,4,5trisphosphate (IP,) has been shown to catalyse the mobilization of Ca? from intracellular stores [ 3 3 ] As . an increased and sustained production of inositol polyphosphates can be observed following rus-expression [ 251, one might expect reduced or depleted intracellular Ca? stores. The depletion of the intracellular Ca?+ stores in Ha-rusexpressing cells should result in a desensitization of the intracellular Ca?+-mobilizing system. In fact, such an Ha-rus-induced desensitization of the Ca? -releasing system stimulated serum growth factor has indeed been observed 1251. Thus the data described so far are in accordance with the working hypothesis that Ha-rus causes a Constitutive activation of growth factor signalling pathways. However, is there any evidence that the observed effect o n intracellular Ca'+ mobilization is caused by the presumed mcchanism? As outlined abovc, it seemed quite likely that this phenomenon is the result of the elevated IP,-formation. However, the experimental data revealed that alteration in inositol polyphosphate formation in MMTV-LTR-Ha-rus-transfected cells cannot be detected before 8 h after addition of glucocorticoid [25I. On the other hand, the desensitization of the Ca"-releasing system to bombesin or serum growth factors is clearly expressed as early as 2 h after induction of p2 1 r'' by steroid hormone 1251. Furthermore, determination o f the Ca?+ load of non-mitochondria1 stores revealed no difference between Ha-rus-cxprcssing versus non-expressing cells 1341. Thus, the Ha-rus-induced dcscnsitization o f the Ca?+-releasing system t o growth factors must be due to another mechanism. Several authors have described alterations in the number or activity of growth factor receptors in rus-transfected cells [35,361. However, down-regulation of receptors could be eliminated as being responsible for the Ha-rus-mediated desensitization of the Ca2+-releasing system to bombesin. We followed the activity of the bombesin receptor by measuring IP3 formation after addition +




of bombesin to quiescent 3T3-cells. The 1P3 response t o bombesin is unchanged up to 6 h after expression of Ha-rus. At this time the desensitization of the Ca? mobilizing system is already maximal 125, 341. A depression o f the bombesin-induced lP3 formation by Ha-rus does occur, but it is a relatively late phenomenon which can be seen only 24 h after expression of the oncogene [34]. Thus this effect is clearly not responsible for the Ca? desensitization immediately following the expression of Ha-rus. Protein kinase C-mediated negative feedback loops could also be eliminated as a cause of the effect on the Ca"-releasing system in Ha-rus-transfected cells ( K . Maly, unpublished work). In view of the available data. a direct interference o f Ha-rus with the IP, receptor seems t o be the most likely mechanism responsible for the desensitization phenomcnon. v-mos has been found to exert identical effects o n the Ca' +-releasing system as Ha-rus [25]. In summary, the two cytosolic oncogene products p2 I "I' and ~ 3 7 " " " affect the Na+/H +-antiporter and the intracellular Ca? -releasing system - two mechanisms involved in growth factor signal transduction. The growth factorindependent activation of the N i l + /H +-antiporter in particular indicates a constitutive stimulation o f ;I mitogenic signalling pathway by these oncogencs. New aspects with regard to the biological function of p2 1'"' were revealed by the discovery o f a rus-specific GTPase activating protein (GAP). p2 1-GTP encoded by transforming rus-oncogenes proved to be insensitive to G A P 1371 (for a review see 1381). These data provide additional support for the notion indicating a continuously activated uncontrolled signal output as a consequence of oncogenic mutations in rus genes. +



Nircleur sigtiul truti.sdiictioti mechunisms The final steps in growth factor signal transduction occur in the nucleus and lead to an activation o f DNA replication. These steps seem t o require transcriptional activation of genes coding for transcriptional factors like the c-fbs, c-myc, and the c-jitn proto-oncogenes (for reviews see [ 39, 401). Induction of p2 1r('' has been shown to enhance transcription of jirri-B and c-jitti, thus providing a link between the membrane-associated and intranuclear oncogene products 14 I]. The detailed mechanisms by which mitogenic signals are transduced to the nucleus arc unknown. Recent evidence from our laboratory indicates that an activation o f histone acetylation may be involved. Post-transitional histone acetylation has been related to the regulation of transcription [42, 431. Recently, it has been shown that there is an additional replication-asociatcd type of acetylation immediately preceding the onset of S-phase following partial hepatectomy [44], after stimulation of quiescent fibroblasts by serum growth factors [45], or following expression o f c-rizyc [45]. Similar observations have been described for I'hysurzrm polycephulitm, in which a wave o f histone acetylation precedes entry into S-phase 1461. Histonc acetylation may be required for a labilization of the binding of histone to DNA and a subsequent destabilization of the nuclcosome as a prerequisite for the onset o f replication. A systematic investigation o f histone acetyltransferase activity in hepatomas o f slow, intermediate and high growth rates revealed elevated activities o f this enzyme in all tumours, suggesting that the increased activity of histone acetyltransferase represents a transformation-linked phenomenon 1471. I . Weinberg, K.A. ( 1985) Sciiwcc, 230, 770-776 2. Goustin, A. S., Leof, E. B., Shipley, G. I). & Moses, H . L. ( 1986) Cancer Res. 46. 1 0 15- I 0 I9 3. Downward, J., Yarden, Y., Mayes, E., Scrace, G., Totty, N., Stockwell, P., Ullrich, A,, Schlessinger, J . & Waterfield, M. D. ( 1984) Nature (London) 3 0 7 , 5 2 1-527



METABOLIC CONTROL IN CANCER 4. Ullrich. A.. Coussens, L.. Hayflick. J. S.. Dull. T. J.. Gray. A.. Tam. A. W.. Lee, J.. Yarden. Y.. Libermann, 'I. A.. Schlessinger. J.. Downward. J.. Mayes. E. L. V.. Whittle, N.. Waterfield. M. D. & Seebzrg, P. H. ( 1984)Nirture (London) 309,4I X-425 5 . Yamamoto. T..Nishida, T., Miajima. N., Kawai, S., Ooi, 1.. & Toyoshima. K. ( 1983)Cell (Cumbridge. Muss.) 35.7 1 -7X 6 . Hunter, I.( 19x7)in Oncogenes und Growth Firctors (Bradshaw, R. R. & Prentis. S., eds.). pp. 135-142.Elscvicr. Amsterdam, New York. Oxford 7. Khashayarsha. K., Dull, T. J., Graf.T., Schlcssingcr. J., Ullrich. A., Beug. H. & Vennstrom, B. ( 1988)EMHOJ. 7,3061-307I X. Rettenmeir, C. W.. Sacca. R., Roussel. M. -1.. Look, A. I. & Stanley. E.R. ( 1985)

Signal transduction mechanisms in cancer.

METABOLIC CONTROL I N CANCER 67 Metbolic Control in Cancer Regulation in Metabolism Group Colloquium Edited by K. Snell (University of Surrey) and O...
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