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Abstract Type 1 insulin-like growth factor (IGF-I) has been implicated in cellular transformation and the acquisition of an invasive/metastatic phenotype in various tumors. A number of strategies have been developed to block this pathway including the identification of molecules able to reduce the production of IGF-I and IGF receptor antagonists. Researchers from McGill University have patented a further way of inhibiting IGF-related tumor growth. Following ligand binding, the IGF-I receptor is internalized and the ligand is separated from the receptor by endosomal proteinases. Blocking this process by cysteine proteinase, or more specifically cathepsin, inhibitors or by cathepsin antisense prevented IGF-I-induced DNA synthesis; anchorage independent growth and MMP-2 synthesis in vitro and metastasis in vivo. According to pharmaceutical databases, few cysteine proteinase inhibitors are reported as being in development suggesting that this target represents an exciting yet under-exploited potential anti-cancer therapy. This research group has developed much of the screening architecture required to exploit this target and licensees or industrial partners are now sought to expedite development.

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Background:

The IGF-IR mitogenic signaling pathway is still being elucidated but a good diagrammatic overview of the sequence of events can be seen at Biocarta. The activity of IGF-I and IGF-2 is mediated in part through the association of receptor tyrosine kinase with Shc, Grb2, and Sos-1 to activate ras and the Map kinase cascade (see inset). This pathway can also be activated by insulin receptor substrate-1 (IRS-1). An endpoint of the Map kinase pathway is modification of transcription factor activity, such as activation of ELK transcription factors. Inhibition of apoptosis is also initiated by tyrosine phosphorylation of substrates which modulate proapoptotic members of the Bcl-2 family (O'Connor et al, 2000).

IGF-IR receptors have been implicated in a variety of regulatory mechanisms in the adult including CNS control of metabolic homeostasis (Unger & Betz, 1998), neuronal survival and protection (see review by Garcia-Segura et al, 2000) and also renal function. Consequently exogenous IGF-I has been reported to protect against cerebral ischemia (Liu et al, 2001) and speed recovery from kidney tubule necrosis (eg Tsao et al, 1997). Considering its powerful mitogenic role, it is hardly surprising that IGF-I also plays a role in cardiovascular remodeling and indeed it has been implicated in hyperplastic and hypertrophic responses to hypertension as well as atherosclerosis, post-angioplasty restenosis, wound healing and recovery from myocardial infarction (Delafontaine, 1995). However, perhaps the largest body of data related to IGF-IR concerns its role in tumor progression. As recently reviewed, IGF-IR has multiple roles in the development of cancer including cell survival, motility, invasion, growth potential in secondary organ sites, the induction of angiogenesis, and the acquisition of multiple drug-resistance (Brodt et al, 2001). Since IGF-I appears obligatory for the establishment and maintenance of the transformed phenotype and for tumorigenesis, blockade of IGF-I activity has been proposed as a target for anti-cancer drugs (Rubin & Baserga, 1995) and a number of potential targets have been identified. These include IGF-I binding proteins, most importantly IGFBP-1, -2 and -3, which play a particularly important role in limiting levels of bioactive IGF-I, and the IGF-I receptor itself which not only mediates the activity of IGF-I itself but also contributes to the tumorgenic activity of oncogenes (Werner et al, 2000), a host of other growth factors and mitogenic hormones (see below).

While the IGF system has been implicated in general in the process of tumorogenesis it's role has been particularly well defined in breast cancer (see Surmacz, 2000; Surmacz et al, 1998). In primary breast tumors, the IGF-IR is overexpressed and hyperphosphorylated, which correlates with radio-resistance and tumor recurrence. IGF-I and IGF-II are both able to stimulate the proliferation of MCF-7 cells at nanomolar concentrations (De Leon et al, 1992), an effect that was reduced by IGFBP-1 (Yee et al, 1994), IGFBP-3 (Ricort & Binoux, 2001), IGF-1 antisense (Neuenschwander et al, 1995) and anti-IGF-IR antibody (De Leon et al, 1992). In vitro, the IGF-IR is required for mitogenesis and transformation, and its over-expression or activation counteract effects of various pro-apoptotic treatments and also enhances cell-cell adhesion through e-cadherin (Guvakova & Surmacz, 1997). On the other hand receptor antagonists increase the sensitivity of breast cancer cell lines to doxorubicin and taxol (Beech et al, 2001). Although there is a clear correlation between IGF-IR as well as IGF-I expression and the progression of breast cancer, expression drops in dedifferentiated tumors (Schnarr et al, 2000). This is thought to represent an effect rather than a cause of tumor progression. Despite this drop, IGF-I is still expressed in advanced tumors and this appears to play a crucial role in metastasis with receptor activation causing chemotactic movement toward components of the extracellular matrix (Doerr & Jones, 1996), the expression of MMP-2 (Long et al, 1998) and also up-regulation of uPA activity via the MAP- and PI3-kinase pathways (Dunn et al, 2001). The therapeutic potential related to the blockade of IGF-IR is put firmly in perspective by recent studies showing that highly metastatic estrogen negative cells lost their ability to proliferate and form colonies when transfected with antisense IGF-IR and furthermore these cells formed smaller and less metastatic tumors in mice (Chernicky et al, 2000). Therefore the IGF-IR pathway appears to represent a key element both of initial tumor growth and also metastatic progression at later time points. Consequently, the IGF-IR pathway offers an attractive anti-cancer target (Brodt et al, 2000).

It addition to contributing to tumor progression per se, IGF-IR signaling appears to impinge on the estrogen pathway. There is a strong positive correlation between estrogen receptor and IGF-IR expression in tumor biopsies (Railo et al, 1994), and in fact estrogens induce key elements of the IGF-IR pathway, while anti-estrogens down-regulate IGF-IR signaling (Huynh et al, 1998; Kawamura et al, 1994; Molloy et al, 2000). On the other hand up-regulation of the IGF-IR pathway, either through over-expression of IRS-1 or reduced expression of IGFBP promotes estrogen-independent growth and transformation (Guvakova & Surmacz, 1997) and tamoxifen resistance (McCotter et al, 1996; Parisot et al, 1999). Tamoxifen resistance often signals tumor escape, and maintaining hormonal sensitivity by reversing this phenomenon represents a further attractive consequence of targeting the IGF-IR pathway.

IGF has also been well studied in the context of lung cancer. IGF-I production is considerably higher in non-small cell lung cancers than surrounding tissue and was proposed to be derived from fibroblasts thereby acting in a paracrine fashion on tumor cells (Ankrapp & Bevan, 1993). Liver- and lung-derived IGF-I was suggested to contribute to the metastatic progression of lung cancer to the liver and to the acquisition of a liver-metastasizing potential(Long et al, 1994; 1995; 1998). This activity was reduced by IGFBP-2, a binding protein often expressed in tumors (Reeve et al, 1993), and both anti-IGF-1 antibody and IGF-I antisense were able to block non-small cell lung cancer proliferation and xenograft growth (Zia et al, 1996; Lee et al, 1996). Human squamous cell biopsies showed a particularly distinctive IGF-IR staining contrastive distinctly with IGF-IIR which was only weakly expressed (Kaiser et al, 1993).

Involvement of IGF-I in two of the top 5 most common causes of cancer-related death (see table) supports the pharmaceutical targeting of this pathway. Different strategies have been developed to prevent IGF-IR signaling, including receptor antagonists and antisense mRNA and post-endocytic degradation of the IGF-I/IGF-IR complex (Brodt et al, 2000). Receptor internalization and dissociation has long been regarded as a key event in the regulation of receptor activity (Ceresa & Schmid, 2000). Endosomal dissociation is regulated by proteinases and on the basis of earlier studies into the recycling of other receptors may involve the cysteine proteinase, cathepsin B (Authier et al, 1995; Authier et al, 1999). This is particularly exciting given the expression of cathepsin B by MCF-7 breast cancer cells (Ishibashi et al, 1999). Furthermore, recent reports describe a correlation between cathepsin B expression and tumor invasiveness (Szpaderska & Frankfater, 2001) as well as a poor prognosis for breast (Maguire et al, 1998) and lung (Inoue et al, 1994) cancer patients.

Cathepsin inhibitors as candidate targets for metastatic cancer: McGill University scientists have recently performed a groundbreaking study to determine the role of cathepsin inhibitors in reducing IGF-I mediated functions. For experimental detail the reader is referred to the original publication describing this study (Navab et al, 2001).

 

• DNA synthesis, anchorage-independent growth and MMP-2 expression are critical for malignant transformation and metastatic progression. Each of these parameters was dramatically reduced in H-59 Lewis lung carcinoma cells by the cathepsin B/cathepsin L inhibitor, E-64. Likewise DNA synthesis and anchorage-independent growth were reduced in MCF-7 breast cancer cells. In previous studies a second, but more specific cathepsin B inhibitor, CA074-methy ester also reduced invasive properties (Szpaderska & Frankfater, 2001).
• E-64 abolished IGF-I/IGF-IR dissociation in both H-59 and MCF-7 cells. This was suggested to involve trapping the receptor in endosomes and also maintaining it, and its substrates in a hyper-phosphorylated state, which in turn attenuates the biological consequences of IGF-IR binding. A second, but more specific cathepsin B inhibitor, CA074-methy ester caused an even more dramatic hyper-phosphorylation of IGF-IR.
• Pretreatment of H-59 cells with E-64 almost abolished the ability of these cells to metastasize to the liver in xenograft models (Navab et al, 1997). Although in vitro effects of E-64 appear to be mediated by cathepsin B inhibition, it can only be speculated that anti-metastatic activity of E-64 in vivo is predominantly due to cathepsin B rather than cathepsin L inhibition. These data do however clearly show that cathepsin inhibitors represent an exciting target for metastasis therapy.

Patent position: McGill University have patented the use of endosomal protease inhibitors to target cellular processing of growth factors, their receptors or subsequent signaling pathways leading as a means of blocking growth factor receptor turnover and tumorigenicity (WO01/44464; patent publication date, June 2001). Please note that the patent claim also includes targeting the cathepsins by antisense and covers cathepsins B, H, L and S

Market size: Therapies able to reduce the biological activity of IGF-IR could fall into the proapoptotic market. This market is still in its infancy (see BCC press release) however it will almost certainly grow, and pharmaceutical agents that modulate apoptosis will probably mature by the end of the decade. This total market is expected to reach $510 million by 2005 and may reach the billion-dollar mark by the end of the decade. The current market most likely to accept IGF-IR modulators is perhaps the hormonal therapy market, which is largely polarized towards the treatment of breast and prostate cancer. Current market players are mainly androgen agonists (for prostate cancer), estrogen antagonists (first line treatment for invasive breast cancer), aromatase inhibitors and LHRH agonists (both second line treatments for invasive breast cancer although there have been recent moves to support the inclusion of aromatase inhibitors as first line breast cancer treatments). As a general guide to market potential, sales of estrogen antagonists amount to $250 million and $350 million in the US and the rest of the world respectively.