The role of insulin receptors and IGF-I receptors in cancer and other diseases

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The role of insulin receptors and IGF-I receptors incancer and other diseasesFrancesco Frasca a; Giuseppe Pandini a; Laura Sciacca a; Vincenzo Pezzino b;Sebastiano Squatrito a; Antonio Belfiore c; Riccardo Vigneri aa Department of Internal Medicine, Endocrinology Unit, University of Catania,Catania, Italy

b Department of Internal Medicine, University of Catania, Servizio di Diabetologia, Ospedale Cannizzaro, Catania, Italyc Department of Clinical and Experimental Medicine Unit of Endocrinology, University of Catanzaro, Campus loc.Germaneto, v.le Europa, Catanzaro, Italy

First Published: February 2008

To cite this Article: Frasca, Francesco, Pandini, Giuseppe, Sciacca, Laura, Pezzino, Vincenzo, Squatrito,Sebastiano, Belfiore, Antonio and Vigneri, Riccardo (2008) 'The role of insulin receptors and IGF-I receptors in cancerand other diseases', Archives Of Physiology And Biochemistry, 114:1, 23 — 37

To link to this article: DOI: 10.1080/13813450801969715URL: http://dx.doi.org/10.1080/13813450801969715

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REVIEW ARTICLE

The role of insulin receptors and IGF-I receptors in cancer and otherdiseases

FRANCESCO FRASCA1, GIUSEPPE PANDINI1, LAURA SCIACCA1,

VINCENZO PEZZINO2, SEBASTIANO SQUATRITO1, ANTONIO BELFIORE3, &

RICCARDO VIGNERI1

1Department of Internal Medicine, Endocrinology Unit, University of Catania, PO Garibaldi Nesima, Via Palermo 636,

95122 Catania, Italy, 2Department of Internal Medicine, University of Catania, Servizio di Diabetologia, Ospedale

Cannizzaro, Catania, Italy, and 3Department of Clinical and Experimental Medicine Unit of Endocrinology, University of

Catanzaro, Campus loc. Germaneto, v.le Europa, 88100 Catanzaro, Italy

AbstractThere is evidence, both in vitro and in vivo, that receptor tyrosine kinases play a key role in the formation and progression ofhuman cancer. In particular, the insulin-like growth factor receptor (IGF-IR), a tyrosine kinase receptor for IGF-I and IGF-II, has been well documented in cell culture, animal studies, and humans to play a role in malignant transformation,progression, protection from apoptosis, and metastasis.

In addition, the hormone insulin (which is very closely related to the IGFs) and its tyrosine kinase receptor (the IR, whichis very closely related to the IGR-IR) have been documented both in vitro and in vivo to play a key role in cancer biology.Indeed, several epidemiological studies have shown that insulin resistance status, characterized by hyperinsulinaemia, isassociated with an increased risk for a number of malignancies, including carcinomas of the breast, prostate, colon andkidney.

Recent data have elucidated some molecular mechanisms by which IR is involved in cancer. IR is over-expressed in severalhuman malignancies. Interestingly, one of the two IR isoform (IR-A) is especially over-expressed in cancer. IR-A is the IRfoetal isoform and has the peculiar characteristic to bind not only insulin but also IGF-II.

In addition, the IR contributes to formation of hybrid receptors with the IGF-IR (HR). By binding to hybrid receptors,insulin may stimulate specific IGF-IR signalling pathways. Over-expression of IR-A is, therefore, a major mechanism of IGFsystem over-activation in cancer. In this respect, IR-A isoform and hybrid receptors should be regarded as potentialmolecular targets, in addition to IGF-IR, for novel anti-cancer therapy.

These findings may have important implications for both the prevention and treatment of common human malignancies.They underline the concept that hyperinsulinaemia, associated with insulin resistance and obesity, should be treated bychanges in life style and/or pharmacological approaches to avoid an increased risk for cancer. Moreover, native insulin andinsulin analogue administration should be carefully evaluated in terms of the possible increase in cancer risk.

Key words: Insulin receptor isoform, IGF-I, hybrid, tumour progression.

Introduction

In recent years it has become evident that the insulin-

like growth factor (IGF) system plays a permissive

role in cancer development and progression (Khand-

wala et al., 2000; Yu & Rohan, 2000; Valentinis &

Baserga 2001; LeRoith & Roberts Jr, 2003; Pollak

et al., 2004). Deregulation of the IGF system is

a common event in several malignancies and

includes IGF-I receptor (IGF-IR) over-expression,

over-activation and autocrine/paracrine production

of IGF-IR ligands, IGF-I and IGF-II (Khandwala

et al., 2000; Yu & Rohan, 2000; Valentinis &

Baserga 2001; LeRoith & Roberts Jr, 2003;

Pollak et al., 2004). Therefore, several investigators

have focused their efforts in developing strategies to

inhibit the IGF-I receptor (IGF-IR) activity in

cancer. The focus of this review is the emerging role

of the insulin receptor (IR) as an important regulator

of the IGF system in cancer.

Correspondence: Francesco Frasca, M.D., Ph.D., Department of Internal Medicine, Endocrinology Unit, University of Catania, PO Garibaldi Nesima, Via

Palermo 636, 95122 Catania, Italy. Tel: þ39 095 759 8702. Fax: þ39 095 47 2988. E-mail: [email protected]

Received for publication 18 December 2007. Accepted 6 February 2008.

Archives of Physiology and Biochemistry, February 2008; 114(1): 23 – 37

ISSN 1381-3455 print/ISSN 1744-4160 online ª 2008 Informa UK Ltd.

DOI: 10.1080/13813450801969715

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IR is structurally homolog to the IGF-IR and is

expressed at high levels in adult muscle, adipose

tissue and liver, where it regulates glucose metabo-

lism in response to insulin. These target tissues of

insulin mainly express one of the two IR isoforms,

IR-B. The second isoform, IR-A, which is obtained

by exon 11 skipping, is present in these tissues at a

lower relative abundance (Mosthaf et al., 1990).

Interestingly, cancer cells overexpress IR at levels

higher than those present in muscle or liver (Frittitta

et al., 1993; Mathieu et al., 1997). Moreover, cancer

cells mostly show an increased capacity to skip IR

exon 11 and to express IR-A, which is the pre-

dominant IR isoform in foetal life (Frasca et al.,

1999). Cancer cells thus acquire a remarkable ability

to respond to circulating insulin, especially when it is

abnormally high as in obese and diabetic patients.

These studies are in agreement with epidemiological

studies demonstrating that obesity and insulin

resistance, which are characterized by hyperinsuline-

mia, are associated with an increased risk of

malignancy, including breast, prostate, colon and

kidney carcinomas (Bray, 2002; Calle & Thun, 2004;

Vigneri et al., 2006). Moreover, IR-A has been found

to be a receptor for IGF-II, which binds with similar

affinity both to IGF-IR and IR-A (Frasca et al.,

1999). Given the close homology between IR and

IGF-IR, hybrid IR/IGF-IR receptors (HR) are

normally formed by random assembly of receptor

hemidimers (Soos et al., 1990, 1993a; Pandini et al.,

1999). Overexpression of both IR and IGF-IR in

cancer cells, leads to a HR overexpression as well.

The relative abundance of the two IR isoforms may

affect the binding affinities of HR, thus contributing

to regulate the activity of the IGF system (Soos et al.,

1990, 1993a; Pandini et al., 1999).

A better understanding of the role of IR and IR

gene splicing in cancer has important implications for

cancer prevention measures, which should include

control of insulin resistance and associated hyper-

insulinemia by changes in life style and, in some

cases, pharmacological approaches. Moreover, in

addition to the IGF-IR, both IR-A and HR should

be also considered as molecular targets for novel

anti-cancer therapies, especially for tumours with a

high IR:IGF-IR ratio.

Insulin receptor structure and signalling

The insulin receptor (IR) is a heterotetrameric

protein consisting of two extracellular a-subunits

and two transmembrane b-subunits. The binding of

ligand to the a-subunit of IR stimulates the tyrosine

kinase activity intrinsic to the b-subunit of the

receptor (Ebina et al., 1985a, b; Ullrich et al., 1985,

1986). Extensive studies have indicated that the

ability of the receptor to autophosphorylate and

phosphorylate intracellular substrates is essential for

its mediation of the complex cellular responses to

insulin (Figure 1) (Ebina et al., 1985a, b; Ullrich

et al., 1985, 1986). Structural studies reveal that the

two a-subunits jointly participate in insulin binding

and that the kinase domains in the two b-subunits are

in a juxtaposition that permits transphosphorylation

of one b-subunit by another on specific tyrosine

residues in an activation loop, resulting in the

increased catalytic activity of the kinase (Ebina

et al., 1985a, b; Ullrich et al., 1985, 1986). The

receptor also undergoes autophosphorylation at other

tyrosine residues in the juxtamembrane and intracel-

lular tail (Ottensmeyer et al., 2000). The activated IR

tyrosine kinase phosphorylates several immediate

substrates including insulin receptor substrate pro-

teins (IRS1-4), DOK4, DOK5, SHC, Gab1, Cbl,

APS and signal regulatory protein family (SIRP)

members (Liu & Roth, 1998; Kankazi & Pessin,

2001). Some of these proteins, including IRS and

SHC, are recruited to a juxtamembrane region in the

receptor containing an NPXY motif (Figure 1), while

others, such as APS, bind directly to the activation

loop (Kaburagi et al., 1995; Ceresa & Pessin, 1998;

Biedi et al., 2003; Harrington et al., 2005). Each of

these phosphorylated proteins provides specific dock-

ing sites for effectors or adapter proteins, containing

Src homology 2 domains (SH2) that specifically

recognize different phosphotyrosine residues, includ-

ing p85 and Grb2 (Figure 1). Although IRS proteins

share a high degree of homology, their functions are

not redundant. Indeed, studies in knockout mice

indicate that IRS-1 deficiency causes growth retarda-

tion, impaired glucose tolerance but not diabetes,

while IRS-2 causes severe insulin resistance and type

2 diabetes (Tamemoto et al., 1994; Withers et al.,

1998). Upon tyrosine phosphorylation, IRS proteins

interact with the p85 regulatory subunit of PI 3-

kinase, leading to the activation of the enzyme and its

targeting to the plasma membrane (Harrington et al.,

2005) (Figure 1). The enzyme generates the lipid

product phosphatidylinositol 3, 4, 5-triphosphate

(PIP3), which regulates the localization and activity

of numerous proteins. PIP3 is inactivated by depho-

sphorylation by the 30 phosphatase PTEN and 50

phosphatase SHIP2 (Stambolic et al., 1998). PI 3-

Kinase is essential for metabolic effects of insulin, as

blockade of PI 3-Kinase by inhibitors (wortmannin)

and dominant negative constructs completely blocks

the glucose uptake in response to insulin stimulation

(Okada et al., 1994). PIP3 formation in response to

insulin results in the recruitment/activation of pleck-

strin homology (PH) domain-containing proteins,

including enzymes, substrates and adaptor and

cytoskeletal molecules. Among these, PDK1 is very

important, as it phosphorylates and activates several

downstream enzymes including the serine/threonine

kinases Akt (PKB) and protein kinase C (PKC)

(Mora et al., 2004) (Figure 1). In addition, PIP3

directly facilitates Akt activation by mediating its

translocation to the membrane via the PH domain.

PIP3 formation also activates PDK2, which

phosphorylates Ser473 on Akt protein (Figure 1)

24 F. Frasca et al.

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(Sarbassov et al., 2005). PDK2 is also important for

the activation of p70S6Kinase, an enzyme regulating

protein synthesis in response to insulin. An impor-

tant Akt substrate is AS160 (Rab-GTPase activating

protein), which is involved in Glut4 translocation to

the plasma membrane in response to insulin and, as a

consequence, in insulin-stimulated glucose uptake

(Sano et al., 2003; Zeigerer et al., 2004) (Figure 1).

Furthermore, Akt activation mediates important anti

apoptotic functions of insulin and insulin like growth

factors (IGF-I and IGF-II). Indeed, activated Akt

phosphorylates the Bcl-2 family member family BAD

(Datta et al., 1997). Phosphorylated BAD is not able

any more to exert its pro-apoptotic function. Another

molecule important for the anti-apoptotic function of

insulin is FKHR (forkhead in human rabdomyosar-

coma). FKHR, together with the other members of

the family FKHRL1 and AFX, is a transcriptional

enhancer, which targets genes regulating apoptosis

and entry into the cell cycle. FKHR phosphorylation

by activated Akt results in nuclear exclusion and

cytoplasmic retention (Kops et al., 1999; Burgering

& Kops, 2002; Kino et al., 2005). It follows that

FKHR phosphorylation is a mechanism by which

insulin inhibits transcription of pro-apoptotic genes.

In addition to p85, phosphorylated IRS-1 and IRS-2

are able to recruit the Grb2/Sos (son of sevenless)

complex from the cytoplasm to the membrane. This

brings Sos in close proximity to RAS, which catalyses

RAS GTP/GDP exchange. Activation of RAS

recruits and thereby activates RAF kinase. RAF

kinase activates MEK1, which in turn activates MAP

kinase (ERK) (Figure 1), a key enzyme in cell cycle

entry and progression (Ceresa & Pessin, 1998).

Moreover, SHC phosphorylation in response to

insulin is able to activate the same pathway and is

required for sustained MAP kinase activation and a

normal mitogenic response to insulin and insulin like

growth factors (Figure 1) (Sasaoka & Kobayashi,

2000). Several studies have shown that Cbl

recruitment and phosphorylation by IR is a

separate pathway, which is localized in lipid raft

Figure 1. Schematic representation of insulin receptor signalling. Insulin binding leads to receptor autophosphorylation and phosphorylation

of several intracellular substrates including IRS1/4. Phosphorylated IRS-1 recruits Grb2/Sos complex, which triggers the RAS/RAF/MEK/

ERK pathway (on the right). This pathway is mainly involved in mediating the mitogenic effect of insulin and insulin like growth factors

(IGF-I and IGF-II). Recruitment of p85 on IRS-1 and IRS-2 leads to PI-3 kinase activation, and, as a consequence, Akt pathway activation

and Glut4 translocation (on the left). This pathway is mainly involved in mediating the metabolic effects of insulin, including glucose uptake,

glycogen and protein synthesis. Moreover, Akt pathway activation is responsible for the anti-apoptotic effect of insulin, IGF-I and IGF-II.

Insulin receptor in cancer 25

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microdomains and is very important for Glut4

translocation (Baumann et al., 2000). Lipid rafts

are specialized regions of the plasma membrane

enriched in particular lipids and proteins and IR

resides in these microdomains, perhaps through

interaction with the raft protein caveolin (Gustavsson

et al., 1999). Activation of IR in these plasma

membrane subdomains recruits APS protein, which

is an adapter containing three SH2 domain. APS

exists as a homodimer and interacts with three

phosphotyrosines in the activation loop of IR b-

subunit via its SH2 domain, so that each activated IR

binds two APS proteins (Hu et al., 2003). Upon

binding to the IR b-subunit, APS is tyrosine

phosphorylated, resulting in the recruitment of the

SH2 of Cbl (Liu et al., 2002). Recruitment of Cbl

onto the IR/APS complex results in Cbl phosphor-

ylation on three tyrosines. Upon tyrosine phosphor-

ylation, Cbl interacts with the protein CrkII, an SH2/

SH3 containing protein. CrkII binds to specific

phosphorylated tyrosines via its SH2 domain (Liu

et al., 2002). Since CrkII is constitutively associated

with the nucleotide exchange factor C3G via its SH3

domain, insulin stimulation cause the C3G/CrkII

complex to be recruited to lipid raft domains, where

it catalyzes the activation of the small G proteins

TC10a and TC10b (Chiang et al., 2001). TC10

proteins are very important for Glut4 translocation,

since overexpression of a dominant negative TC10

inhibits insulin stimulated glucose uptake.

The insulin receptor is a member of the IGF

system

The IR is highly homolog to the insulin-like growth

factor receptor (IGF-IR). Homology between IR and

IGF-IR ranges from 45–65% in the ligand binding

domains to 60–85% in the tyrosine kinase and

substrate recruitment domains (Ullrich et al., 1986;

Anderson et al., 1995; Mynarcik et al., 1997; Yip

et al., 1988; Whittaker et al., 2001). Both receptors

have evolved by a common ancestor gene and are

part of a system, which is highly conserved in

vertebrates and invertebrates and to co-ordinate

metabolic and growth responses in multicellular

organisms in response to nutrient availability

(Brogiolo et al., 2001; Drakas et al., 2004). Reduced

signalling through the insulin/IGF pathway impairs

growth, by decreasing both cell size and cell number

(Brogiolo et al., 2001; Drakas et al., 2004; Kozma &

Thomas, 2002). Reduced insulin/IGF signalling also

activates cellular stress protective programs that may

contribute to extended lifespan. Reduced insulin

signalling due to starvation induces a developmental

arrest at the stage of a long-lived dauer larva in C.

elegans (Burgering & Kops, 2002; Finch & Ruvkun,

2001; Hafen, 2004).

In mammals, IR and IGF-IR have evolved to exert

different biological functions. The IR has acquired a

central role in glucose homeostasis, while the IGF-IR

has become the regulator of body growth in response

to pituitary growth hormone (GH). One important

basis of this different role is the different cell

distribution of the two receptors: in adult differen-

tiated tissues IR is expressed at high levels only in

adipose tissue, muscle and liver, while IGF-IR is

expressed at significant levels in virtually all tissues

(Moller et al., 1989; Soos et al., 1990; Giddings &

Carnaghi, 1992). Moreover, several studies have

highlighted small differences in the recruitment of

intracellular mediators by either IR or IGF-IR.

Comparison of IR and IGF-IR signalling in trans-

fected NIH-3T3 cells indicated that IR was more

efficient that IGF-IR in activating the IRS-1 pathway

(Mastick et al., 1994). However, the two receptors

were equally potent in activating the Shc/ERK

pathway and DNA synthesis ( Mastick et al., 1994).

Similar results have been obtained in transfected

Rat1 fibroblasts, where IGF-IR was more efficient

than IR in activating the ERK pathway and DNA

synthesis (Sasaoka et al., 1996).

One important difference between IR and IGF-IR

is the ability to induce cell transformation. R- rat

fibroblasts, which are knocked-out for the IGF-IR by

homologous recombination, are refractory to onco-

gene driven cell transformation (Sell et al., 1994). In

those cells, the ability to undergo transformation is

restored by re-expression of IGF-IR and but not of

IR (Sell et al., 1994). Moreover, IGF-IR, but not IR

overexpression in R- cells causes a ligand-dependent

transformed phenotype (Sell et al., 1994). In

contrast, IR overexpression is sufficient to induce a

ligand-dependent phenotype in NIH3T3 fibroblasts,

which express endogenous IGF-IR (Giorgino et al.,

1991). This effect was inhibited by a monoclonal

antibody against the IR with blocking activity

(Giorgino et al., 1991). Similar results where

obtained by IR overexpression in immortalized

human breast epithelial cells 184B5 (Frittitta et al.,

1995). These data suggest that IGF-IR has a more

potent transforming effect than IR, but, in immorta-

lized cells with endogenous IGF-IR, IR overexpres-

sion is sufficient to induce a ligand-dependent

transformed phenotype.

A variety of studies have compared the ability of IR

and IGF-IR to induce differential recruitment of

intracellular substrates and gene expression activa-

tion. For instance, the adapter protein Grb10 and the

membrane protein CEACAM-2 involved in receptor

down-regulation preferentially interact with the IR

(Laviola et al., 1997; Soni et al., 2000). In contrast,

other substrates involved in the regulation of

proliferation, apoptosis, including CrkII, 14-3-3

proteins and IIP-1 preferentially associate with the

IGF-IR (Beitner-Johnson & LeRoith, 1995; Furla-

netto et al., 1997; Ligensa et al., 2001). IR and IGF-

IR can also have an opposite regulatory role. a-5

integrin is up-regulated by IGF-I but down-regulated

by insulin (Lynch et al., 2005). Moreover, while IGF-

IR activation results in FAK (focal adhesion kinase)

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phosphorylation, IR activation causes a FAK de-

phosphorylation (Pillay et al., 1995; Baron et al.,

1998). This opposite effect of IR and IGF-IR on

FAK activation is caused by the preferential activa-

tion of c-Abl tyrosine kinase by IR (Frasca et al.,

2007).

Global gene expression, studied by micro-array

technology, has demonstrated many similarities, but

also some differences, between the two receptors.

Studies have been carried out both in cells trans-

fected with either wild type IR or IGF-IR and in cells

transfected with TrkC/IR or TrkC/IGF-IR chimeric

receptors (Dupont et al., 2001; Mulligan et al.,

2002). Both these studies have found that most

genes are similarly regulated by the two receptors.

However, a subset of genes can be differentially

regulated by the activation of either the IR or the

IGF-IR pathway. Genes responsive to IGF-I were

primarily involved in the regulation of proliferation,

adhesion or differentiation, while genes responsive to

insulin fell in a broader spectrum and could not be

categorized into a particular group. Functional

differences between IR and IGF-IR are especially

evident in postnatal life, whereas they closely

cooperate in regulating cell proliferation in prenatal

life. Genetic data obtained from knockout mice

indicate that both receptors are required for an

optimal embryonic development, while glucose

metabolism seems unimpaired in prenatal life in IR

knock-out mice (Nevado et al., 2006).

Taken together, these results indicate that both IR

and IGF-IR are approximately equally potent in

activating DNA synthesis when that they are equally

expressed. Physiologically, the growth-promoting

role of IR is evident in prenatal life, when the two

receptors are required for embryo development. Data

are insufficient to give a clear picture about the

relative activity of the two receptors regarding other

biological effects, including apoptosis protection and

cell migration. In conclusion, these two receptors

have both common and distinct regulatory effects on

cellular proliferation, differentiation, and morpho-

genesis, all effects that can be potentially relevant in

cancer biology.

The insulin receptor in cancer

It has been known for some time that several cancer

cell lines require insulin for optimal cell growth. This

effect has been commonly attributed to the spillover

of high insulin concentrations on the IGF-IR.

However, the presence of insulin binding sites in

human breast cancer cells in culture was reported

more than 25 years ago (Osborne et al., 1978) and

has been subsequently described in several other

cancer cell lines. Moreover, insulin receptors (IRs)

have also been found in most normal and neoplastic

hematopoietic cells (B-lymphoblasts, T-lympho-

cytes, plasmocytoma cells) where they have a role

in the regulation of proliferation and differentiation.

A murine T-cell lymphoma cell line (LB cells) has

been described, which expresses fairly high amounts

of IR but minimal IGF-IR levels. In these cells

growth is dependent on IR activation by insulin

(Pillemer et al., 1992). Moreover, early studies in the

animal model have suggested a direct role of insulin

in cancer growth. MCF-7 human breast cancer cells

do not form tumours in diabetic nude mice, while

they do form tumours in 100% diabetic nude mice

treated with insulin (Nandi et al., 1995). Conversely,

breast tumours induced by the carcinogen 7, 12

dimethylbenz(a)anthracene (DMBA) in the rat re-

gress when the rats are made diabetic by alloxan

administration, which destroys pancreatic b-cells and

causes insulin deficiency. Administration of exogen-

ous insulin restored tumour growth, while estrogens

were ineffective (Heuson et al., 1972). Ovariectomy

caused regression of DMBA-induced tumours in

some rats but not in all. Interestingly, tumours that

continue to grow in spite of ovariectomy showed

increased insulin binding compared with tumours

that regressed. Similar results were obtained by other

authors in mice made diabetic by the use of

streptozotocin (Cohen & Hilf, 1974). Moreover,

mice transplanted with LB lymphoma cells devel-

oped resistance to lymphoma growth when made

diabetic by streptozotocin treatment or fed a low-

energy diet (Sharon et al., 1993). It is interesting to

note that both conditions were characterized by low

insulin levels. Taken together these studies indicate

that insulin binding sites are present in many

malignant cells and that insulin may be involved in

the growth of these malignancies. However, the

possible implications of these findings for human

cancer remained unclear for long time.

Studies performed by specific ELISAs have in-

dicated that approximately 80% of breast cancers

showed an IR content higher than the mean value

found in normal breast and approximately 20% of

cancers showed IR values over 10 fold higher than

mean value in normal breast (Papa et al., 1990).

Immunostaining indicated that IR was predomi-

nantly overexpressed in neoplastic cells and not in

stromal adipocytes and inflammatory cells. The

binding affinity of IRs was similar in cancer and

normal breast tissues (Frittitta et al., 1993). Func-

tional studies indicated that IR expressed in breast

cancer was more sensitive to insulin than in normal

breast as far as autophosphorylation of the b-subunit

was concerned. Studies on the possible prognostic

significance of IR overexpression indicated that

patients with tumours with high IR content had a

lower 5-year disease-free survival (DFS) than pa-

tients with tumours with moderate IR content

(Mathieu et al., 1997) (Figure 2). Multivariate

analysis of these data, including established prog-

nostic factors, confirmed that IR content was the

strongest independent predictive factor for DFS.

However, IR overexpression is not specific of breast

cancer but seems to be a common phenomenon in

Insulin receptor in cancer 27

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human cancer. Increased IR content was found in

carcinomas of the colon, lung, ovary and thyroid

(Frasca et al., 1999; Frittitta et al., 1999; Vella et al.,

2001, 2002).

Putative mechanisms of IR overexpression in

cancer include the p53 inactivation and overexpres-

sion of HMGA1 proteins. Indeed, p53 suppress IR

and IGF-IR promoter activity (Ohlsson et al., 1998)

and this finding may explain, at least in part, why

IGF-IRs are also overexpressed in most human

carcinomas (Papa et al., 1993; Baserga et al., 1994;

Blakesley et al., 1997; Surmacz, 2000). HMGA1

proteins belong to the family of non-histone chro-

matin proteins and are often referred as architectural

transcription factors because of their ability to

regulate gene expression through DNA binding and

participation to multiprotein complexes (Sgarra et al.,

2004). HMGA1 expression positively correlates with

the transformed/metastatic phenotype (Reeves, 2001;

Sgarra et al., 2004) and, recently, it has been involved

in IR gene transcription regulation (Foti et al., 2003).

Moreover, recent reports indicated that HMGA1

may inhibit p53 family member tumour suppressor

function (Frasca et al., 2006; Pierantoni et al., 2006,

2007), thereby suggesting that abnormally expressed

HMGA1 may up-regulate IR expression in cancer

cells by inactivating p53. Taken together, these data

suggest that IR overexpression is driven by multiple

mechanisms commonly activated in cancer. Some of

these mechanisms also cause IGF-IR overexpression,

although the regulation of IR and IGF-IR may be

partially different.

One relevant issue is to clarify whether insulin

elicits biological effects in cancer cells by acting via

its own receptor or by activating the IGF-IR. Studies

performed with blocking monoclonal antibodies

specific to the IR and IGF-IR have addressed this

issue (Milazzo et al., 1992). In estrogen responsive

breast cancer cell lines (MCF-7, ZR-75 and T47-D),

growth response to insulin could be specifically

blocked by an anti-IR but not by the anti-IGF-IR

blocking antibody (Milazzo et al., 1992). In the same

cell system, an anti-IR stimulating antibody induced

cell growth. These data consistently demonstrated

that the mitogenic effect of insulin in breast cancer

cells is due to insulin binding to its own receptor and

not to the IGF-IR, which has an approximately

100-fold lower affinity. These data are in agreement

with studies carried out in IR transfected cells

(Mamounas et al., 1989; Giorgino et al., 1991;

Mastick et al., 1994). Insulin is also able to stimulate

directional cell motility toward a ligand gradient

(chemotaxis) likewise other growth factors (Benoliel

et al., 1997; Maehiro et al., 1997; Sciacca et al.,

2002). This effect requires IR autophosphorylation

and the activation of the same signalling pathways

involved in mitogenesis. Since cell motility is relevant

to tumour metastases, breast cancer cells overexpres-

sing IRs may have an increased metastatic potential.

Substitutions of one or more amino acid residues

of insulin by recombinant DNA technology has

allowed the creation of insulin analogues able to

improve glycaemic control in diabetes. Studies with

insulin analogues further support the direct role of

insulin and IR in cancer cells (Ish-Shalom et al.,

1997; Shymko et al., 1999) and shed light onto the

role of insulin in mitogenesis. Since some of these

analogues bind to the IR with a low dissociation rate

and form receptor-ligand complexes with increased

half-life, this abnormal binding property confers to

these insulin analogues an enhanced mitogenic

potency. This effect may be explained by the

prolonged IR and Shc phosphorylation (Ish-Shalom

et al., 1997; Shymko et al., 1999; De Meyts &

Shymko, 2000). Similarly, breast cancer cells have an

impaired insulin receptor down-regulation in re-

sponse to insulin and an increased half-life of

receptor-insulin complex (Mountjoy et al., 1987).

Evidence of the transforming potential of the insulin

analogues are also available in vivo: treatment with a

low Kd insulin analogue AspB10 induces tumours of

the mammary gland in female rats and, after a 24-

month treatment, 44% rats developed benign breast

diseases and 23% developed breast cancer (Drejer,

1992). These results are in accordance with data

obtained in vitro indicating that AspB10-insulin

causes phenotypic changes in non-transformed hu-

man breast epithelial cells (Milazzo et al., 1997).

Taken together, these evidences strongly suggest a

role of IR activation in tumour progression.

Insulin receptor isoforms

The human insulin receptor (IR) is encoded by a

single gene, which is located on chromosome 19 and

contains 22 exons. The mature IR exists as two

isoforms, IR-A and IR-B, which result from the

Figure 2. Effect of insulin receptor expression on disease-free

survival for breast cancer. Disease-free survival in patients operated

for node-negative breast cancers positive for IR expression at

immuno-histochemistry. The subset of patients with tumours

showing high IR expression had a shorter disease-free survival than

patients with moderate IR expression (Mathieu et al., 1997).

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alternative splicing of the primary transcript. The IR-

B differs from IR-A by the inclusion of exon 11,

which encodes a 12 amino acid fragment (residues

717–728) of the IR a-subunit. Inclusion of this exon

is differently regulated in various tissues and diseases

(Figure 3). Indeed, while IR-A is ubiquitously

expressed, IR-B is predominantly expressed in tissue

targets of insulin metabolic effects, including liver,

muscle, adipocytes and kidney (Moller et al., 1989;

Mosthaf et al., 1990). IR-A is mainly expressed in

foetal tissue and is up-regulated in several diseases

including type 2 diabetes, cancer and myotonic

dystrophy (Denley et al., 2003). Currently, the

mechanisms underlying IR splicing are poorly under-

stood. Transient transfection experiments in HepG2

cells with minigenes spanning from exon 10 to 12

have allowed the identification of the important

sequences for the splicing process. Indeed, a 48-

nucleotide purine-rich sequence at the 50 end of

intron 10 functions as a splicing enhancer and causes

an increase in exon 11 inclusion (Kosaki et al., 1998).

Moreover, a 43-nucleotide sequence, favouring

skipping of exon 11 has been mapped upstream of

the branch point sequence of intron 10. Mutations in

exon 11 have also indicated the existence of exon 11

sequences playing an active role in determining the

degree of exon inclusion in both a positive and

negative manner. Further minigene analysis indi-

cated that sequences in exon 10, exon 11 and exon

12 are responsible for the splicing process, maybe

because they are recognized by specific splicing

factors including U1 snRNP, SF1 and U2AF65/35;

in particular, strengthening of either the 50 or 30 splice

sites in exon 11 by mutagenesis leads to its

constitutive inclusion. In contrast, strengthening of

upstream and downstream splice donor and acceptor

sites on the neighbouring exons (10 and 12) leads to

a decreased exon 11 inclusion. The specific splicing

factors regulating exon 11 skipping or inclusion are

difficult to identify (Webster et al., 2004). Indeed,

overexpression of particular splicing factors by

transfection did not provide encouraging results

because these proteins are already maximally ex-

pressed in cultured cells. However, indirect evidence

is available showing that overexpression of SF2/ASF

may promote inclusion of alternatively spliced exons

in the rat clathrin light chain B and rat b-tropomyo-

sin (Mayeda et al., 1993; Caceres et al., 1994). This

effect is due to the ability of SF2/ASF to promote the

use of a proximal splice site, either 50 or 30 over a

distal site (Mayeda et al., 1993; Caceres et al., 1994).

More interestingly, this activity is antagonized by the

hnRNP-A1 splicing factor, which favours the use of

distal splice sites over proximal. Hence, the observed

choice of splice sites reflects a balance between SF2/

ASF and hnRNP-A1. These results are in accor-

dance with the observation that cancer cells, which

express mostly IR-A, overexpress also hnRNP-A1

splicing factor (Zerbe et al., 2004; Ushigome et al.,

2005). More recently, another possible IR splicing

factor has been identified in Mytonic Dystrophy type

1 (DM1) patients, whose skeletal muscle tissues

predominantly express IR-A, rather than IR-B

(Savkur et al., 2001, 2004). In these patients,

CUG-BP, a regulator of pre-mRNA splicing, was

up-regulated and overexpression of CUG-BP in

normal cells induced a switch to IR-A (Ho et al.,

2005). Further studies in DM1 indicated that the

effect of CUG-BP on IR splicing is not direct, but

mediated by the muscle-blind-like proteins

(MBLN1), which promote the inclusion of exon

11. Indeed, depletion of MBLN1 by siRNA results in

increased exclusion of exon 11 (Dansithong et al.,

2005). Additional studies have shown that MBLN

proteins are antagonized by embryonic lethal abnor-

mal vision-type RNA-binding protein 3-like factors

(CELFs), which promote exon 11 exclusion (Ladd

et al., 2004; Ho et al., 2004). These results suggest

that a balance between MBLN and CELFs may

modulate the fine-tuning of IR isoform expression in

cells and tissues. Moreover, recent studies, per-

formed in rat skeletal muscle myotubes, indicate that

insulin itself is able to enhance exon inclusion in pre-

mRNA for protein kinase CbII (PKCbII)1. This

effect of insulin occurs by phosphorylation of the

SRp40 splicing factor in Akt2 dependent manner

(Patel et al., 2001, 2004, 2005). Since IR exon 11

inclusion occurs mainly in tissues targets of insulin

action, it is reasonable to hypothesize that insulin the

major regulator of the shift from IR-A to IR-B in

these differentiated tissues.

Figure 3. Schematic representation of IR splicing, isoform

expression and function. The insulin receptor gene is located on

chromosome 19 and contains 22 exons. Inclusion or exclusion of

exon 11 in IR mRNA leads to two different IR isoforms: IR-A

(exon 11-) and IR-B (exon 11þ). Exon 11 inclusion is regulated by

SF2/ASF and MLBNs, while exon 11 exclusion is favoured by

hnRNP-A1 and the CELF family of splicing factors. IR-B is mainly

expressed in tissue targets of the metabolic effects of insulin. IR-A

is expressed in foetal, cancer tissue, CNS and hematopoietic cells.

While IR-B binds only insulin, IR-A may bind both insulin and

insulin-like growth factor II (IGF-II). In this view, IR-A may

mediate some of the IGF-II effects on cell proliferation in cancer

and foetal development.

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The IR-A and IR-B display several functional

differences: IR-A has a twofold higher affinity for

insulin, a faster internalization and recycling time, an

overall lower signalling capacity and a twofold lower

tyrosine kinase activity (McClain 1991; Yamaguchi

et al., 1993). Indeed, a switch from IR-A to IR-B

induced by dexamethasone in hepatoma cells corre-

lated with increased insulin sensitivity (Kosaki &

Webster, 1993). In addition, in pancreatic b-cells

insulin stimulation of either IR-A or IR-B elicits

different effects by the preferential activation of

different phosphatidylinositol-3 kinase and protein

kinase isoforms (Leibiger et al., 2001; Uhles et al.,

2003). Data obtained in murine 32D hematopoietic

cells indicate that IR-A preferentially triggers mito-

genic, and anti-apoptotic signals, whereas IR-B cell

differentiation signals (Sciacca et al., 2003). More

recent evidence indicates that IR-A, but not IR-B

was able to fully restore glucose uptake in hepato-

cytes from IR knockout (IRKO) mice (Nevado et al.,

2006).

Of major interest is the finding that the IR-A, but

not the adult IR-B, is activated by IGF-II at high

affinity (Frasca et al., 1999) (Figure 3). This

important difference of the two IR isoforms was

observed by transfecting either IR-A or IR-B cDNAs

in R-mouse fibroblasts, which are IGF-IR gene

deficient by homologous recombination. In the

absence of interference of IGF-IR, the binding

affinity of IGF-II for IR-A for was very high

(ED50¼ 3.0+ 0.4 nM) similar to that observed with

the classical IGF-IR (1.6+ 0.3 nM). These findings

were also obtained in transfected NIH-3T3 cells

and CHO cells, indicating that it is a general

phenomenon and not dependent on a cell type

(Frasca et al., 1999).

Effects of IGF-II and insulin, studied in R-cells

transfected with the IR-A, indicated that IGF-II is

more effective than insulin in stimulating cell

proliferation, while insulin is more potent than

IGF-II in stimulating glucose uptake (Frasca et al.,

1999). Similar data were also found by other authors

(Morrione et al., 1997). When signalling pathways

were analyzed in R-/IR-A cells, quantitative and

temporal differences in the phosphorylation of

intracellular substrates were observed in response to

insulin or IGF-II. In particular, both the IRS/PI3K

and the Shc/ERK pathways were less intensely and

more transiently activated after IGF-II than after

insulin stimulation (Frasca et al., 1999). However,

the peak of activation in terms of protein phosphor-

ylation of Shc and ERK1/2 were much less affected

than peak of activation of IRS-1/2 and PI3K, leading

to a relative preponderance of the activation of the

first pathway after IGF-II stimulation (Frasca et al.,

1999). In SKUT-1 leiomyosarcoma cells, which lack

functional IGF-IR, results were even clearer: insulin

was more potent that IGF-II in stimulating the PI3K/

Akt pathway and in inhibiting cell apoptosis, while

IGF-II was more potent than insulin in activating the

Shc/ERK pathway (Sciacca et al., 2002) and in

stimulating cell chemo-invasion (Sciacca et al.,

2002). These observations raised the possibility that

IGF-II, by acting on IR-A, may be more effective

than that can be predicted by its affinity to the

receptor.

To address the post-receptor events differentially

activated by insulin and IGF-II via IR-A, micro-array

studies in R-/IR-A cells were performed (Pandini

et al., 2004): while 214 transcripts were similarly

regulated by insulin and IGF-II, only 45 genes were

differentially affected. In more detail, eighteen of

these differentially regulated genes were exclusively

responsive to one of the two ligands (12 to insulin

and 6 to IGF-II). These data, showing that IGF-II is

more potent than insulin in the induction of certain

genes, are in line with previous ones showing that

IGF-II was more potent than insulin in stimulating

mitogenesis and cell migration. These data provide a

molecular basis for understanding the biological role

of IR-A in embryonic/foetal growth and the selective

biological advantage for malignant cells producing

IGF-II and expressing IR-A.

Relative expression of the two insulin receptor

mRNA transcripts is regulated in a tissue-specific

manner: hematopoietic and neuronal cells express

only the IR-A; tissues such as placenta, kidney,

adipose tissue, and skeletal muscle express both

isoforms; liver predominantly expresses IR-B. The

IR isoform expression is also development-specific:

IR-A is predominantly expressed in human foetal

tissues including kidney, skeletal muscle, liver, and

fibroblasts (Frasca et al., 1999). Indeed, IR-A

expression in foetal cells is very important for

embryonic development, as it may mediate the

growth promoting effect of IGF-II. Analysis of

mouse dwarfing phenotypes resulting from targeted

mutagenesis of the IGF-I and IGF-II genes and the

cognate IR and IGF-IR gene (Liu et al., 1993; Louvi

et al., 1997) indicate that embryos lacking both IGF-

IR and IGF-II are more severely growth-retarded

than single IGF-IR knockout mice (Liu et al., 1993;

Louvi et al., 1997), suggesting that IGF-II may act

also via another receptor, which could be IR-A.

Accordingly, the phenotype of double IGF-IR/IGF-

II knockout mice is similar to that of double IGF-IR/

IR knockout mice, thereby suggesting that IR

mediates some of the IGF-II effects (Liu et al.,

1993; Louvi et al., 1997). Taken together these

results suggest that while IR-B is a receptor for the

metabolic effects of insulin, IR-A is a receptor

involved in mediating the mitogenic effect of IGF-

II in embryonic life.

Several lines of evidence indicate that IR isoform

expression may be regulated by hormones: RT-PCR

analysis performed in human hepatoblastoma cell

line HepG2 indicated a predominant IR-A expres-

sion and exposure to thyroxine and dexamethasone,

caused a predominant expression of IR-B (Kosaki &

Webster, 1993). Similar results were also obtained in

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rat preadipocytes, which express predominantly IR-

A. Exposure of these cells to adipocyte differentiation

medium, which contains insulin and dexamethasone,

led to the appearance of IR-B (Serrano et al., 2005).

The alteration of the relative expression of IR-A

and IR-B has been also reported in diseases like type

2 diabetes and insulin resistance: in particular, the up

regulation of IR-A in diabetic patients, which is

twofold less efficient in mediating insulin signalling

than IR-B, may partially account for the insulin

resistance status (Benecke et al., 1992; Norgren et al.,

1993; Hansen et al., 1993; Huang et al., 1994; Sesti

et al., 1995; Sbraccia et al., 1996). This hypothesis

has been recently reinforced by results obtained in

Mytonic Dystrophy type 1 (DM1) patients. These

subjects display a significant insulin resistance, which

is accompanied by an increased relative expression of

IR-A in muscle in comparison with normal subjects

(Savkur et al., 2004). Taken together, this evidence

implicates DM1 as a model for studying IR splicing

mechanisms and the role of IR aberrant splicing in

human metabolic diseases like type 2 diabetes and

insulin resistance.

Overall, these data suggest that IR splicing gen-

erates two receptors with different role and functions:

while IR-B is the classical receptor for metabolic

effects of insulin in muscle, liver and adipose tissues,

IR-A is a receptor for IGF-II, which may mediate the

growth promoting and anti-apoptotic effects of these

growth factors under physiological conditions like

embryonic development. The regulation of the IR-A/

IR-B ratio in various tissues and cells may have

important consequences for responsiveness to both

insulin and IGF-II and may be deranged in diseases

such as type two diabetes, Mytonic Dystrophy type 1,

and cancer. Moreover, the fine regulation of IR-A/

IR-B ratio expression may be rendered more com-

plex by the co-expression of the cognate IGF-IR,

which may form hybrid receptors with IR.

Insulin receptor isoforms and cancer

At variance with IGF-IR, the biological role of IR

overexpression in cancer was difficult to explain,

since insulin is not locally produced like IGF-I and

IGF-II. However, approximately 40% of unselected

human breast carcinomas express levels of IR higher

than IGF-IR, indicating that IR overexpression may

actually confer a selective advantage to cancer cells

(Papa et al., 1990). A key observation to clarify this

issue was the finding that IR of breast cancer cells,

but not of normal breast cells could be activated not

only by insulin but also by IGF-II (Sciacca et al.,

1999). In contrast, no major difference was observed

in the affinity of insulin binding to IR between

normal and cancer tissues. The evidence that

autocrine IGF-II could sustain cell proliferation by

IR activation was obtained in MDA-MB-157 breast

cancer cells, which produce IGF-II in an autocrine

fashion and express 5-fold more IR than IGF-IR. In

these cells, the use of anti-IR antibody MA-51

inhibited IGF-II stimulated proliferation more effec-

tively than the anti-IGF-IR antibody (Sciacca et al.,

1999). These results confirmed the experimental

hypothesis that the IR overexpression may be

important in mediating IGF-II biological effects.

The general relevance of these data in human breast

cancer was obtained in both breast cancer cell lines

and breast cancer tissue specimens, which mainly

express IR-A, while IR-B is predominant in normal

breast cell lines and tissues (Sciacca et al., 1999).

Many other malignancies, including carcinomas of

the colon, lung, ovary, thyroid and myosarcomas

were also found to predominantly express IR-A

rather than IR-B (Frasca et al., 1999; Sciacca et al.,

1999, 2002; Vella et al., 2002; Kalli et al., 2002;

Denley et al., 2003). This phenomenon, concomitant

with IR overexpression, leads to the expression of

very high levels of IR-A. With respect to thyroid

cancer, IR-A overexpression and autocrine IGF-II

production are clearly linked to tumour progression

and de-differentiation. Indeed, both IR and IGF-IR

increase during thyroid tumour progression, but only

IR expression further increases in poorly differen-

tiated and anaplastic thyroid carcinomas (Vella et al.,

2002). Moreover, the relative IR-A abundance and

autocrine IGF-II production in thyroid cancer follow

the same trend. As observed in breast cancer, the

biological effects of IGF-II in thyroid cancer cell lines

are predominantly mediated by IR-A, as IR-A

blockade by a specific antibody is able to inhibit the

the proliferative effect of IGF-II ( Sciacca et al., 1999,

2002; Vella et al., 2002). IR-A overexpression in

cancer may be interpreted in light of the fact that the

IGF-IR mediates not only proliferation and apoptosis

protection, but also cell differentiation. This function

is associated with specific region of the carboxy-

terminus not shared by the IR-A. The IR-A and the

IR-B may also play a different role in cell differ-

entitation: transfection of 32D cells with IR-A, but

not with IR-B, impairs cell differentitation (Sciacca

et al., 2003). IR-A overexpression was also found in

other malignancies, including ovarian cancer and

myosarcomas where it is also present a significant

IGF-II expression (Sciacca et al., 2002; Kalli et al.,

2002).

Hybrid insulin/IGF-I receptors (HRs)

The mature IR is formed by two hemireceptors (each

composed of one a and one b subunit), linked by

disulfide bonds at the level of the Fn0 and Fn1

extracellular domains (Yip, 1992) (Figure 4). In cells

and tissues expressing both IR and IGF-IR, IR

hemireceptors may heterodimerize with IGF-IR

hemireceptors, leading to the formation of hybrid

IR/IGF-IRs (HRs) (Soos et al., 1990, 1993a, b;

Yamaguchi et al., 1993; Whittaker et al., 1990). IR/

IGF-IR heterodimerization is made possible by the

high degree of homology shared by the two receptors,

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which ranges from 41 to 67% at the level of

extracellular domains, and is approximately 45% at

the level of Fn0 and the Fn1 domains (Yip, 1992).

The physiological role of HRs is not entirely clarified,

because studies on HRs are complicated by the

concomitant expression in cells and tissues of a

variable amount of both IR and IGF-IR and

antibodies specific for HRs are not fully available

(Soos et al., 1990, 1993a, b; Yamaguchi et al., 1993;

Whittaker et al., 1990). Moreover, purified HRs,

obtained by affinity chromatography, may be con-

taminated with homodimeric IR or IGF-IR that have

reduced functional activity. However, studies carried

out with purified hybrid receptors indicate that these

receptors mostly bind IGF-I, while they bind insulin

with a much lower affinity (Soos et al., 1993b;

Pandini et al., 1999) (Figure 4). In accordance with

the high affinity for IGF-I, HR-s have been measured

as the proportion of total IGF-I binding sites that

may be immunoprecipitated with an anti-IR anti-

body. This method indicated that in normal tissues

HRs represent 40-90% of total IGF-I binding sites

(Soos et al., 1993a). Studies performed in transfected

cells have shown that, HR formation occurs by

random assembly of hemidimers. According to this

model, the expected proportion of HRs can be

calculated as follows: HRs¼ 2�IR�IGF-IR (where

HRs, IR and IGF-IR are the number of binding sites

per cell) (Siddle et al., 1994). However, the modula-

tion of the assembly of either homodimeric receptors

or HRs by unknown factors cannot be excluded.

Since cancer tissues express abnormally high levels

of both IR and IGF-IR, the HR content is

particularly elevated, as assessed by direct ELISA

performed in a variety of human cancer cells and

tissues. Breast and thyroid carcinomas were the most

extensively studied and, in both these tumours, HRs

exceeded IGF-IR content in most cases (Pandini

et al., 1999; Belfiore et al., 1999). HRs appear to play

an important role in mediating IGF-I effect in breast

and thyroid cancer cells: in cells expressing more

HRs than IGF-IRs, IGF-I mitogenic effect is more

strongly inhibited by anti HR blocking antibody than

anti-IGF-IR antibody. The opposite is true when

cancer cells express more IGF-IRs than HRs

(Pandini et al., 1999; Belfiore et al., 1999).

Further studies were designed to evaluate HRs

with respect to IR-A and IR-B. Experiments per-

formed in transfected cells indicated that both IR-A

and IR-B can form hybrids with IGF-IR with the

same efficiency (HR-A and HR-B), in close accor-

dance with the random assembly model (Pandini

et al., 2007). This observation indicates that the

relative abundance of HR-A (containing IR-A) or

HR-B (containing IR-B) simply depends on the

relative abundance of the two IR isoforms (Figure 5).

Taken together, these data indicate that cancer cells

mostly overexpress HR-As. One immediate practical

consequence of these studies is that HRs should be

taken into account by therapies designed to target

IGF-I effects in cancer.

HR is a broad specificity receptor for ligands

of the IGF family

As HR-As are overexpressed and functional in cancer

cells, which are often exposed to high levels of

autocrine/paracrine IGF-I/II, it is of interest to

understand their ligand specificity and signalling

capacity. To answer these questions R- cells were

transfected with IGF-IR and with either IR-A or IR-

B. Cell clones expressing similar amounts of either

HR-A or HR-B were used for binding studies.

Interestingly, HR-A binds IGF-I, IGF-II and insulin,

while, HR-B mostly binds IGF-I (Pandini et al.,

2002). Remarkably, IGF-I bound HR-A with higher

affinity than HR-B. These data were confirmed in

HepG2 human hepatoblastoma cells. These cells,

which predominantly express IR-A (approximately

80% of total IR), can be switched to express IR-B by

treatment with dexamethasone. Autophosphoryla-

tion of purified HRs in response to the different

ligands reflected binding data, indicating that HR-A

is a functional receptor for IGF-I, IGF-II and insulin,

Figure 4. Schematic representation of hybrid receptors (HR). In

cells and tissues expressing both IR and IGF-IR, IR hemi-

receptors may heterodimerize with IGF-IR hemi-receptors,

leading to the formation of hybrid IR/IGF-IRs (HRs), which

bind IGF-I and IGF-II with high affinity and insulin with a much

lower affinity.

Figure 5. Schematic representation of HR-A and HR-B. In cells

and tissues expressing either insulin receptor isoform A (IR-A) or

B (IR-B) and IGF-IR, IR hemi-receptors may heterodimerize with

IGF-IR hemi-receptors, leading to the formation of either hybrids

containing IR-A (HR-A) or IR-B (HR-B).

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while HR-B is more selective for IGF-I. As a

consequence, cells expressing HR-A are more

sensitive to biological effects of both IGFs and

insulin, such as proliferation and migration, as

compared to cells transfected with HR-B (Pandini

et al., 2002, 2007) (Figure 6). The intracellular

signalling of HRs is not completely elucidated.

However, there is evidence suggesting that b-subunit

moieties, belonging to both IR and IGF-IR, are

phosphorylated in HRs. Interestingly, insulin binding

to HR-A is able to activate the b-subunit of the IGF-

IR moiety, and, as a consequence, both IGF-I and

insulin are able to induce phosphorylation of Crk-II,

a specific substrate of IGF-IR (Pandini et al., 2002).

These data indicate, therefore, that insulin binding to

HR-A may provide a way for insulin to effectively

cross-talk with the signalling capability of IGF-IR as

insulin binding affinity for HR-A (ED50¼ 4.5 nM

insulin for 125I-IGF-I displacement) is much higher

than its affinity for IGF-IR (ED504 1000 nM

insulin). These effects may be particularly

relevant in foetal and cancer tissues, where IR-A is

expressed at high levels and/or in the presence

of hyperinsulinemia (obesity, insulin resistance,

type 2 diabetes). In contrast, the predominant IR-B

expression occurring in normal differentiated cells

reduces cell sensitivity to IGFs by causing sequestra-

tion of IGF-IR moieties into low affinity HR-B

(Figure 6).

Relevance of IR-A overexpression and HR-A

in cancer prevention and treatment

IR-A overexpression in cancer sensitizes cancer

cells to autocrine IGF-II and also to circulating

insulin, especially when insulin levels are chronically

high. At the same time, it leads to increased

formation of HR-A, which seems to have unique

binding and functional characteristics. Therefore

IR-A overexpression in cancer has practical implica-

tions both in cancer prevention and treatment.

Implications in cancer prevention

Recent data indicates an increased frequency of

cardiovascular diseases and malignancies in devel-

oped countries because of changes in life style

characterized by reduced physical activity and

increased dietary intake (Sherman et al., 1981;

Folsom et al., 1990; Adami et al., 1991; Bruning

et al., 1992; Bianchini et al., 2002; Vigneri et al.,

2006). These changes have caused an epidemic of

obesity and metabolic abnormalities, including the

so-called ‘‘metabolic syndrome’’, which are charac-

terized by insulin resistance and compensatory

hyperinsulinemia (Bruning et al., 1992; Lev-Ran,

1998; Reaven, 2001; Giovannucci, 2001). However,

insulin resistance associated with obesity or meta-

bolic syndrome is essentially restricted to glucose

metabolism, while the insulin effect on cell prolifera-

tion is relatively unimpaired (Ma et al., 2004).

Compensatory hyperinsulinemia is, therefore, com-

monly associated with increased proliferation of

ovarian techal cells and keratinocytes, causing poly-

cystic ovary syndrome and acanthosis nigricans,

respectively (Apridonidze et al., 2005). Circulating

insulin levels in these patients reach 0.2-0.6 nmol/L,

concentrations similar to those required to induce a

half maximal IR autophosphorylation in breast

cancer specimens (Frittitta et al., 1993). Several

recent studies have reported an association between

abdominal obesity and/or insulin resistance and

increased risk for a variety of malignancies, including

carcinomas of the breast, colon-rectus, endome-

trium, stomach and prostate (Schapira, 1991; Bray,

2002; Bianchini et al., 2002; Vainio et al., 2002; Calle

et al., 2003; Calle & Kaaks, 2004; Calle & Thun,

2004). Other studies have shown that both type 2

Figure 6. Schematic diagram of different ligand and receptor subtypes of the IGF system in physiological and pathological conditions. IR-A

and HR-A, which are especially increased in cancer and poorly differentiated tissues, represent receptors with broad ligand specificity and

have the final effect of up-regulating the IGF signalling pathway.

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diabetes and impaired glucose tolerance, conditions

characterized by insulin resistance and hyperinsuli-

nemia are also associated with an increased risk of

cancer. Hyperinsulinemia in type 2 diabetes may be

worsened by the administration of insulin secretago-

gues (e.g. sulfonylureas) or exogeneous insulin

(Bruning et al., 1992; Bianchini et al., 2002; Hafen,

2004; Coughlin et al., 2004; Evans et al., 2005;

Bowker et al., 2006). Accordingly, some authors have

found that the risk of cancer is increased by treatment

with both sulfonylureas and insulin, whereas is

actually decreased by treatment with metformin, an

anti-diabetic drug which reduces insulin resistance.

Finally, insulin resistance is associated with advanced

cancer disease at diagnosis and increased recurrence

rate. Hyperinsulinemia may both directly stimulate

IR-A and HR-A, which are overexpressed in cancer

cells, and increase IGF-I bioavailability by suppres-

sing the levels of IGF binding proteins 1 and 2 (IGF-

BP1 and IGF-BP2) (Wang & Wang, 2003; Lukanova

et al., 2004; Jenab et al., 2007). Moreover, insulin and

IGFs synergize with thyroid stimulating hormone

(TSH) and sex steroids and have a role in promoting

thyroid and steroid-sensitive tumours. Prevention

and treatment of obesity and insulin resistance by

dietary and lifestyle changes and, when appropriate,

with the use of insulin sensitizers, should, therefore be

considered for cancer prevention.

Implications in anti-diabetic drug development

The different ability of IR and IGF-IR to regulate cell

growth and glucose metabolism together with their

different cell distribution are the basis of active drug

development in the fields of diabetes and cancer. The

difficulty of mimicking the normal insulin secretion

profile in diabetic patients by administering exogen-

ous insulin is well known (Dineen et al., 1995;

Kubota et al., 1996; Saudek, 1997). This has led to

an exploration of the possibility of modifying the

kinetics of insulin by introducing substitutions of one

or more amino acid residues by recombinant DNA

technology. Many companies have, therefore, devel-

oped insulin analogues with a shorter or a longer

half-life that can be advantageously used to improve

glycemic control in diabetes (Ebeling et al., 1996; Le

Roith, 2007). However, it is now evident that these

insulin analogues should be accurately tested for

their ability to stimulate IR-A and/or HR-A. The

study of some early insulin analogues have shown

that they may bind to the IR with a low dissociation

rate, thus forming receptor-ligand complexes with

increased half-life causing prolonged IR and Shc

phosphorylation. This abnormal binding property

confers an abnormally high mitogenic:metabolic

ratio on these insulin analogues. In particular, a low

Kd insulin analogue (AspB10) is able to cause

phenotypic changes in non-transformed breast cells

in vitro and to induce both benign and malignant

tumours of the mammary gland in female rats in vivo

(Milazzo et al., 1997). The study of the mitogenic:

metabolic ratio is now a prerequisite for considering

any new insulin analogue for therapeutical use.

However, other biological effects, such as the effect

on chemotaxis are equally important. In addition, the

study of biological effects may be fairly insensitive

and may be critically dependent on cell context.

Moreover, since even subtle differences in receptor

affinity and activation kinetics may differentially

activate gene expression and biological effects after

IR-A binding, it is reasonable to suggest that gene

expression analysis should also be part of the

program when comparing insulin analogues with

regular insulin.

Implications in anticancer drug development

Drugs aiming at IGF-IR inhibition are able to inhibit

cancer growth and, especially, metastatic spread

(Jones et al., 2004; Knowlden et al., 2005; Camirand

et al., 2005; Catrina et al., 2005; Warshamana-

Greene et al., 2005; Hopfner et al., 2006; Nakayama

et al., 2006; Morgillo et al., 2007; Tazzari et al.,

2007). In this regard we should consider that some

malignancies may have a high IR-A:IGF-IR ratio

(Figure 6). In these conditions, most of the IGF-IR

moieties combine with IR-A moieties to form HR-A.

Therefore, IGF-I will mostly signal through HR-A

(and not IGF-IR), while IGF-II will mostly signal

through IR-A. Therefore, in order to block the IGF-I

signal, we need antibodies that recognize both anti-

IGF-IR and HR-A (anti-IGF-IR antibodies may not

bind to HR-A) (Pandini et al., 2007). In tumours

with a high IR-A:IGF-IR ratio, a combination of

antibodies, including antibodies recognizing HR-A

and IR-A, should possibly be more effective than

antibodies simply blocking homodimeric IGF-IR.

Concluding remarks

Recent findings about the effect of IR gene splicing

on the IGF system may be summarized as follows:

(1) The foetal IR isoform, IR-A, is activated by

both insulin and IGF-II.

(2) IR-A/IGF-II interaction results in a unique

signal, which is different from that provided

by IR-A/insulin interaction and by IGF-IR/

IGF-II interaction.

(3) It is not clear how this signal exactly impacts

tumour biology, although there is some evi-

dence to suggest that it is associated with cell

dedifferentiation.

(4) The relative abundance of IR-A or IR-B affects

the cell responsiveness to insulin and IGFs

through HR formation.

(5) HR-A has a high affinity and broad specificity

for all three ligands of the IGF family, while

HR-B is specific for IGF-I and characterized by

a reduced affinity.

34 F. Frasca et al.

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IR-A and HR-A overexpression in tumour cells

provides a mechanistic explanation for epidemiolo-

gical studies showing an association between insulin

resistance and compensatory hyperinsulinemia and

increased risk of cancer development or poor cancer

prognosis. These findings should reinforce the

recommendation to prevent and, when present, to

aggressively treat all conditions associated with

insulin resistance. As far as the association between

type 2 diabetes mellitus and cancer is concerned,

these studies suggest to prefer the use of insulin

sensitizers over sulfanylureas or insulin, whenever

possible. Newly developed insulin analogues should

be rigorously tested for their affinity with IR isoform

and HR subtypes. With regard to cancer therapy,

these findings on the role of IR-A in the regulation of

the IGF system, suggest that, in addition to IGF-IR,

both IR-A and HRs should also be considered as a

target, especially in tumours that have a high IR-A/

IGF-IR ratio.

Acknowledgements

We thank the American Italian Cancer Foundation

(AICF) for a fellowship to Giuseppe Pandini and

Associazione Italiana Ricerca sul Cancro (AIRC) for

a grant to Riccardo Vigneri. These studies were

also supported by grants from MIUR PRIN 2006

(University of Catania) to Vincenzo Pezzino and

Sebastiano Squatrito.

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