The Role of Insulin-Like Growth Factor System in Soft Tissue Sarcomas: From Physiopathology to Targeted Therapeutic Approaches

Purpose/Results. Although surgical, chemo- and radiotherapeutic treatment regimens in patients with soft tissue sarcomas have constantly been refined over the past two decades, the survival rate for these patients is rather low. Discussion. There is a great need to investigate the mechanisms for oncogenesis and to identify the factors involved in malignant transformation in sarcomas. Among these factors, IGFs are thought to play a pivotal role as progression factors in various types of sarcomas. The dysregulation of the IGF-II synthesis, e.g. by loss of imprinting which occurs in most types of sarcomas, is a permissive effect through the suppression of cell death. In addition, cells that overexpress the type I IGF receptors are more susceptible to transformation by oncogenes. As TP53 suppresses the activity of IGF-II P3 and P4, as well as the type I IGF receptor promoter, mutations of TP53 in sarcomas may alternatively lead to the activation of these factors. Finally, the phenomenon of non-islet cell tumour hypoglycaemia that occurs in patients with sarcomas, and which is related to the secretion of IGF-II prohormones, is discussed. Future therapeutic strategies may be based upon the application of antibodies or antisense oligonucleotides directed against the type I IGF receptors, with the common goal of inducing apoptosis in sarcoma cells. Ultimately, these and other therapeutic approaches may lead to a better outcome in patients suffering from sarcoma.


Introduction
Steady progress has been m ade in the identi® cation of genetic alterations and prognostic factors in sarcom as. However, survival rates of patients with sarcom as remain unsatisfac torily low in spite of aggressive treatm ent involving highly toxic m ultidrug chemotherapeutic as well as radiotherapeutic regim ens. H owever, considerable progress has been m ade in the past decade in identifying im portant factors involved in tumour growth. In addition, the factors involved in the induction and m odulation of apoptosis, am ong them IG Fs, are of particular interest. IGF-I and IGF-II are m itogenic polypeptides and are synthesized by m ost norm al and m alignant tissues, where they are involved in autocrine and/or paracrine types of action. T he IGF system is further com plicated by the presence of speci® c IGF -binding proteins (IG FBP-1 to 2 7) which bind IG Fs with high af® nity. These IG FBPs are thought to play an im portant role in m odulating IG F responsiveness in norm al as well as m alignant cells. The biological effects of IGF s are effected through the insulin, type I IGF (IG F-I) and type II (IGF-II) receptors. T he type I IGF receptor in particular is involved in growth, differentiation and inhibition of apoptosis depending on activation by IGF s. 1 Rescue from cell death by IG F-I is m ediated through this receptor, and antibodies against this receptor can block the rescuing function of the growth factor. 2 M oreover, IGF-I had no effect in preventing etoposide-induced apoptosis in ® broblasts derived from m ice em bryos that have a targeted disruption of the type I IG F receptor. 3 The autocrine growth hypothesis states that both norm al and m alignant cells can synthesize and secrete polypeptide growth factors that will bind to their own cell surface receptor and stim ulate cell proliferation. 4 IG Fs are abnorm ally expressed in som e paediatric solid tumours, and certain tum ours are responsive or dependent upon IGFs for proliferation. In addition, m any tum our cell lines express IGFs as autocrine factors and IGFBPs, w hich, in turn, regulate the bioavailability and bioactivity of IGF s. 5± 7 T he imprinted genes IG F-II and H19 are expressed during embryonal life and are downregulated postnatally. IG F-II is upregulated in paediatric tum ours and developm ental syndrom es predisposing to such tumours (e.g. Beckw ith± W iedemann syndrom e). These factors represent tum our m arkers as they display a tissue-spe ci® c oncofetal pattern of expression. 8 T ransgenic mice overexpressing IG F-II have a higher risk of developing tum ours, including sarcom a, after a long latent period, which suggests that IG F-II functions prim arily as a tum our prom oter or progression factor in vivo rather than a potent tumour initiator. 9,10 Understanding the biology of these growth factors and their receptors can lead to new therapeutic approaches.

D iscussion
T he evalu ation of 29 hum an sarcom a specim en s revealed high levels of expression for IG F -I, IG F -II and the type I IG F receptor m R N A , as d eterm ined by R T -PC R an d in com parison w ith control cell lin es. 11 A nalysis of soft tissue sarcom a cells revealed high steady-state levels of the type I IG F receptor m R N A transcripts and protein w hich correlated w ith receptor-spe ci® c tyrosine kinase activity. 12 H ypophysectom y profound ly in hibits the m etastatic behavior of injected RIF -I ® brosarcom a cells in m ice, w hereas G H adm inistration provokes the o ccu rrence of lun g m etastasis, and it w as conclud ed that som atostatin analogues, G H or IG F antagonists m ay suppress m etastasis of certain tum ou rs. 13 T he presence of IG F -I m R N A in leiom yom as an d leiom yosarcom as, 14 and the IG F -I im m unoreactivity in leiom yosarcom as (7 ou t of 8), synovial sarcom as (2/3), liposarcom as (3/6), ® brosarcom as (1/3) and in one an giosarcom a, 15 su ggest the potential role for IG F -I in stim ulating cell proliferation in these tum ou rs. C o ntrary to the u niform pattern o f IG F -I im m unoreactivity seen in other sarcom as, on ly the spin dle cell and not the epithelial com ponen t of syn ovial sarcom as exhibited strong IG F -I im m u noreactivity. In m align ant ® brous histiocytom as, heterogeneous stain in g w as observed for IG F -I, m ostly seen as a diffu se cytoplasm atic reaction in the m ajority of the tum ou r cells. A separate study in tw o nonin¯am m atory ® brous histiocytom as show ed also sign i® cant imm u nohistochem ical stain ing for IG F -I. 16 A bund ant IG F -II m R N A species have also been detected in histiocytom a tissue. 1 7

Leiomyom as and leiom yosarcom as
In n orm al m yom etrium and leiom yom as, the IG F-II gene is expressed at low levels but it is activated in leiom yosarcom as, w hereas the IG F-I gen e appears repressed in leio m yosarcom as. 1 8 T he expression of a fou rth leader exon (h E4 ) w hich lead s to the form ation of a 5.0 -kb m RN A is en hanced 10-fo ld in leiom yosarcom a tissu e, but it has yet to be established w hether there is a cau sal relation betw een the activation of hE 4 expression and tum our form ation . 19 In norm al sm ooth m uscle and in leiom yom as the IG F-II gen e appeared to be m ethylated , w hereas, in leiom yosarcom as, m ethylation of D N A w as low and it w as suggested that there is an in verse correlation betw een the m ethylation state and expression of the IG F-II gen e. 20 R elaxation o f IG F-II gen om ic im printing w as also observed in uterine leiom yosarcom ata. 2 1 T here w as also a correlation betw een an A vaII restriction fra gm ent len gth polym orphism in the IG F-II gene and the occu rren ce of sm ooth m u scle tum ou rs. 22 M ore than 90% of lipo sarcom as exhibit greatly elevated IG F -II m R N A levels, w hile n orm al adipose tissue contain ed very low or u nd etectable IG F-II levels. 23 Ewing sarcom a/primitive neuroectodermal tum our M ost neuroectoderm al tumour cell lines and tum ours w ith a t(11;22) translocation (prim itive neuroectoderm al tumour (PN ET ), Ew ing' s sarcoma, esthesioneuroblastom a) expressed IGF -I m RN A, whereas none of the cell lines w ithout the translocation (PN ET, neuroblastom a) expressed IGF -I m RN A transcripts. 24,25 D ata indicate that IGF -I m ay play an im portant role for the growth of ES/ PNE T tumour cells. 26 Loss of im printing (LO I) of IG F-II occurs in som e Ew ing' s sarcom as but is not associated with increased expression of IG F-II m RN A, suggesting that LO I m ay be related to genetic or epigenetic abnorm alities in tumours independent of IG F-II expression. 27 The IG F-I receptor-m ediated loop was found to be constantly present in ES/PN ET cells and the addition of a speci® c type I IG F receptor (a IR-3) antibody suppressed the growth of ES/PN ET cells by decreasing the proliferative rate and increasing apoptosis. Furtherm ore, the a IR-3 antibody signi® cantly inhibited the ability of ES/PN ET cells to grow in soft agar and m igrate following a chem otactic stim ulus. 28

Rhabdom yosarcom a
Recent evidence links abnorm al developm ent of the skeletal m uscle pathway with rhabdom yosarcom a. T he shh/ptc/pax signaling pathway is involved in the induction of m yogenic differentiation in somites and in neural tube tissue. A consistant feature of alveolar rhabdom yosarcom a is a translocation involving either a fusion of Pax-3 or, to a lesser extent, Pax-7 w ith another transcription factor fkhr. 45 Binding of shh to ptc activates the zinc ® nger transcription factor gli-1 , which is frequently ampli® ed in hum an sarcom as and brain tum ours. 46 M ice heterozygous for ptc inactivation show a high incidence of rhabdom yosarcom a with overexpression of ptc, gli-1 and IG F-II in the tum our, but not in surrounding norm al skeletal tissue. T his suggests a cross-talk between the ptc and IG F-II signaling pathways in the pathogenesis of rhabdom yosarcom a. 47 M ost rhabdom yosarcom as possess two or m ore copies of active IG F-II alleles, arising either by relaxation of im printing or duplication of the active allele, whereas in norm al muscle m onoallelic expression of the IG F-II gene is conserved. 48 Of the four RM S heterozygotes, 50% had biallelic expression of IG F-II. 49 F urtherm ore, the imprinting of all IG F-II promoters is relaxed in RM S, indicating that loss of im printing of IG F-II gene prom oters m ay be regulated in a coordinated m anner by a com m on m echanism in these tumours. 50 In em bryonal RM S, a tum our-su ppressor locus has been im plicated at chrom osome band 11p15.5. 51 Furtherm ore, there is evidence that this tumour suppressor is im printed in a m anner opposite to that of IG F-II. 52 M atsum oto et al. 53 concluded that loss of im printing of IG F-II itself m ight not induce tum our occurrence in tissues where the control of tissue-speci® c expression of IG F-II is m aintained, and that increased expression of IG F-II due to m aternal loss of a putative controller gene for IGF-II at 11p15 m ight predispose to sustaining tum origenic m utations and tum our progression, and that loss of a putative onco-suppressor gene at 11p15 m ight induce RM S occurrence. H19 is another possib le tum our-suppressor gene for embryonal rhab dom yosarcom a as it is also located at chrom osome 11p15.5 and is paternally im printed. 54,55 Expression of H19 was observed in four out of six embryonal rhabdom yosarcom as. 56 T he expression of the H19 gene is signi® cantly suppressed as com pared to norm al m uscle tissue in 13 out of 15 rhabdom yosarcom as with embryonal histology, and in three out of 11 rhabdom yosarcom as classi® ed as alveolar subtype. It is evident that the genetic and epigenetic alterations affecting chrom osome 11p15 in a high num ber of RM Ss cause deregulation of several im printed genes, including the extinction of H19 and an increase in the num ber of active IGF-II alleles, thus eventually leading to tum our growth. 57 On the contrary, cellular growth rates are reduced in H19 transfected embryonal rhabdom yosarcoma cell line. 58 Rhabdom yosarcom a, a tum our of skeletal m uscle origin, app ears developm entally arrested at an early stage in the m yogenic differentiation pathw ay. IG F-II m RN A has previously been shown to be expressed at high levels in RMS. 59,60 In the hum an RM S cell line IN 157, high levels of 6.0-, 4.8-and 4.2-kb IGF -II m RN A transcripts are expressed, whereas norm al skeletal m uscle expresses negligible am ounts of IGF-II m RN A. 61 In addition, this cell line secretes two forms of IG F-II of m edium m olecular size, 10 and 7.5 kD a, which play a role in the autocrine control of cell growth. 62 A m inor 4.8-kb m RN A was exclusively engaged in the synthesis of the prepropeptide on m em brane-bound polysom es, w hereas a m ajor 6.0-kb m RN A w as present in a cytoplasm atic particle, suggesting that translational discrim ination betw een the m RN As is dictated by their different 59 -untranslated regions. 63 High levels of IGF-II m RN A are found in both alveolar and em bryonal RM S. 64± 66 IGF -II m RN A expression is lim ited to tum our cells and is not found in the surrounding strom a, suggesting that an autocrine loop for IG F-II m ay be functional in vivo in RM S. 65 Poorly differentiated RM S showed the highest level of IG F-II m RN A expression, whereas well-differentiated RM S show ed low expression, albeit still signi® cantly higher than in norm al differentiated skeletal m uscle ® bers. T hus, IGF-II has potential as a m arker for rhab dom yosarcom as and other soft tissue sarcom as that could be especially useful in differential diagnosis of these tum ours. 67 Furtherm ore, IGF-II overexpression in m yoblasts resulted in an increased proliferative rate, im pairm ent of the ability to differentiate into m yoblasts and acquisition of the capability of anchorage-independent growth. It was concluded that IG F-II overexpression in m uscle m yoblasts leads to m orphological and biological changes typical of the m alignant phenotype, and represents a pivotal event in the pathogenesis of RM S. 68 The capacity of growth factors to induce a m otility response in cells has im portant implications for the invasive and m etastatic potential of tumour cells in particular. IGF s, in particular, have the capacity to stim ulate cellular m otility in tum our cell lines via different receptors and probably different signal transduction pathways. As a consequence, enhanced m otility confers upon the cell an increased m etastatic potential. 7 Exogenous IG F-II stim ulates cellular m otility of rhab dom yosarcom a cell lines via an IGF -I receptor-independent pathway. 69 IG F-II elicits a m itogenic response through the type I IGF receptor and a m otility response through the type II IGF receptor. 70 Suram in is a polysulfonated naph thyl-urea with antineoplastic activity which interferes non-selectively with the binding of growth factors to their cellular receptors. In particular, it displaces [ 125 I] IGF -I from the type I IGF receptor, indicating that suram in exerts its effect on RM S cell growth by interrupting the IG F-II autocrine loop in these cells. 71 In the alveolar RM Z-R C2 and the em bryonal CC A RMS cells, suram in induces a signi® cant increase in the proportion of m yosin-positive cells over control cultures. T hus, suram in both inhibits growth and induces myogenic differentiation. 72 In RD and H TB114 RMS cell lines w hich express m utant T P53 protein, transfection with w ild-type TP53 expression vectors led to a reduction in IG F-II P3 prom oter expression in these cells. 73 Furtherm ore, TP53 binds to the P4 proxim al prom oter element which results in the inhibition of P4 activity together with a 5-fo ld reduction of IGF-II in m RN A derived from the P4 prom oter. 74 In RMS cells, tum our-derived form s of T P53 stimulated the activity of the type I IGF receptor, suggesting that wild-type T P53 has the potential to suppress the type I IG F receptor in the differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. 75 Alveolar rhabdom yosarcom a cell lines are very sensitive to the growth-inhibiting effects of the imm unosuppressive agent, rapam ycin, which inhibits the type I IGF receptor-m ediated signalling. 76 A speci® c type I IG F receptor-blocking antibody (a IR-3) suppresses RM S growth in vivo. T he decrease in tum our growth was asso ciated with a decrease of p34 cdc2 , which is involved in cell cycle regulation suggesting that treatment results in the arrest of cellular proliferation. 77 Transfection of a hum an alveolar RM S cell line with an am pli® able type I IGF receptor antisense expression vector w as asso ciated with m arkedly reduced growth rates in in vitro, im paired colony form ation in soft agar and a failure of tumour form ation in im m unode® cient m ice. 78 T he Rh30 alveolar RM S cells, which are stably transfected with antisense type I IG F receptor, show ed signi® cant reduction in growth rate, an increased expression of M yoD , myosin heavy chain, and an increased num ber of m ultinucleated cells in com pariso n to the parental line. The expression of a type I IG F receptor that carries a m utation in the intracellular b -subunit markedly decreased the response of RM S cells to stim ulation with IGF-I and, in addition, resulted in a decrease of RM S growth in vivo suggesting that a prospective gene therapy m ay use this novel strategy to inhibit RMS growth. 79 In addition, recombinant hum an a 2a-in terferon induced growth arrest in these cells, asso ciated with dow n-regulation of the type I IG F receptor. 80 Overexpression of type I IG F receptors and/or IG F ligands m ay thus confer a proliferative advantage on sarcom as over norm al adjacent tissues. T reatm ent of both embryonal and alveolar hum an RM S cell lines with all-trans-retinoic acid resulted in a dosedependent inhibition of cell growth which is not reversed by addition of exogenous IG F-II. 81 M olecular therapies m ay also target the signal transduction pathway, in particular the SH C-G RB2 protein com plexes and M AP kinases w hich have been characterized in RM S tum ours and cell lines. 82 Increased circulating levels of IG FBP-2 were found in various neoplastic conditions, including W ilm s' tum ours 83 and it was concluded that IGFBP-2 measurem ents might be of value as a m arker for m onitoring tum our patients during therapy. Serum IG FBP-2 levels were increased in patients w ith solid peripheral tum ours, whereas patients in com plete rem ission had norm al IGFBP-2 levels. 84 In culture, the A 673 RM S cell line has been shown to secrete a speci® c IG FBP found also in the spinal¯uid. 85 In m edia conditioned by RM S cell lines A 673 and RD , leiom yosarcom a cell line SK-LM S, as well as leiom yoblastom a cell line G 402, IG FBP-2 has been found in large amounts. 86 W hereas IGFBP-3 are synthesized at high levels by the leiom yoblastoma cell line G 402, 86 levels were decreased in leiom yosarcom a sections 87 w hich m ay confer a growth advantage upon m alignant sm ooth m uscle cells.

N on-islet cell tum our hypoglycaemia
Sarcom as are occasionally associated with the occurrence of hypo glycaem ia. N on-islet cell tumours w hich induce hypo glycaem ia are rare. T hey are usually intra-abd ominal or thoracic. T he underlying m echanism for the hypo glycaem ia is the production of IGF-II, predom inantly as a high-m olecular w eight form (`big' IG F-II), by these tum ours. 88,89 T he IG F-II gene is overexpressed in m any m esenchym al tum ours, and the levels of`big' IGF -II are increased in serum from patients with non-islet cell tum our hypo glycaem ia (NIC TH). 90 In patients with haem angiopericytom a, hypoglycaem ia was asso ciated with increased serum levels of`big' IGF -II. 91,92 T he serum of a patient w ith a large intra-abd ominal haem angiopericytom a contained m ainly a largem olecular weight precursor IGF-II (m ol. wt. 15± 20 kD a), which disappe ared from the serum after operation. 93 In another patient with a m eningeal haem angiopericytom a and a large m etastatic liver, hypoglycaem ia was asso ciated with low insulin, distinctly decreased IG F-I and norm al IGF-II, but high`big' IGF-II levels. 94 High levels of IG F-II m RN A and IG F-II peptide w ere detected in both prim ary m eningeal haem angiopericytom a and m etastatic foci in the liver. 95 In a patient w ith pelvic clear cell sarcom a, severe hyp oglycaem ia linked to elevated production of`big' IG F-II with acrom egaloid sw elling returned to norm al after tumour resection. 96 A patient with a huge ® brosarcom a in the right liver lobe, associated with hypo glycaem ia, becam e euglycaem ic after transcatheter arterial embolization. Interestingly, IGF -II intensely stained in the G olgi area of the tumour cells. 97 In a leiom yosarcom a, high concentrations of IG F-II m RN A and elevated IG F-II im munoreactivity were detected with a 77% fraction of high-m olecular w eight IG F-II 98 and, in a histiocytoma, a 100-fo ld elevated IGF -II m RN A level was found as compared to norm al liver. 99 Thus, low or norm al IG F-II levels are found in serum , despite dem onstrable overexpression of IG F-II m RN A by the tum our. 100 It was suggested that abnorm al IGF -II binding to the 150-kD a IGF BP m ay play a role in tum our-associated hypo glycaem ia. 101 The pro-IG F-II is, to a large extent, bound to low-molecular w eight IGF-BPs which are able to freely exit the vascular compartm ent and reach target tissues, w here the IG F-II m ay exert its insulin-like activity. 102 Serum IGFBP-3 was also expressed in form s of about 60 kD a instead of the expected size of about 140 kD a. 103 Furtherm ore, not only suppression of IG FBP-3 but also a 10-fold increase of IG FBP-2 levels has been described. 104

Conclusion
T here is form idable evidence w hich supports the notion that IGFs play a pivotal role in hum an cancer. Relaxation of IG F-II genom ic im printing occurs in childhood as w ell as adult-onset tum ours, and m ay thus represent a novel epigenetic m echanism for oncogenesis throughout life. T he overexpression of the type I IG F receptor renders the cells susceptible to transfo rm ation by oncogenes. T hus, attempts are currently being made to inhibit cell proliferation by targeting the IGF -I receptor, by m eans of anti-receptor antibodies, IGF analogues or antisense strategies, with the com m on goal of inducing apoptosis of neoplastic cells. 105 These therapeutic possib ilities do offer an intruiging scenario to be developed further in an effort to provide patients affected w ith different kinds of soft tissue sarcom as with a better outcom e.

A cknowledgem ent
I w ish to express m y gratitude to D r. Paul N . Scho® eld , D epartm ent of A natom y, U niversity of C am bridge, E nglan d, for his kind su pport and stim ulating discu ssions. T he support of the K ind erkrebshilfe H eid elberg is also gratefully acknow ledged .