Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-02-16
2002-05-07
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007200, C435S007210, C435S320100, C435S325000, C436S063000, C436S064000
Reexamination Certificate
active
06383733
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of tumour therapy.
BACKGROUND OF THE INVENTION
More than 80% of tumours occurring in man are of epithelial origin. The formation of epithelial tumours (carcinomas) is a multi-stage process which is illustrated most clearly in the progression of human colon carcinoma (Powell, et al., 1993) and skin tumours in mice (Wright, et al., 1994). Carcinomas are assumed to start from individual cells or small groups of cells in which mutations have occurred. These cells develop into benign, epithelial hyper- or dysplastic regions. The progression of these hyperplastic regions into a carcinoma in situ, which may then acquire invasive and metastatic properties, requires a number of further mutations in the tumour cell. Characteristically these cells acquire the ability to break down their basal membrane proteolytically, to develop from a stationary polarised cell into a non-polarised cell capable of migrating in the tissue, to survive in the bloodstream and form metastases at remote sites (Liotta, et al., 1991; Liotta and Stetler-Stevenson, 1991).
Although deep changes in gene expression are involved in the many changes in the architecture and behaviour of malignantly transformed cells, none of these newly acquired properties occurs only in invasive tumour cells. Attachment to the basal membrane, proteolysis thereof and migration through the basal membrane and the underlying mesenchyme are important stages in normal processes, e.g. in the implantation of the trophoblast, in movements of configuration during development of the embryo, in the development of the mammary gland and the reorganisation of epithelia during wound healing (Aznavoorian et al., 1993).
For a better understanding of the development and progression of carcinomas it is crucial to understand how the deregulation of these normal processes takes place in cell invasion and metastasisation.
Studies in recent years have contributed to an understanding of the molecular mechanisms involved in the modulation of the epithelial phenotype in normal and pathological situations (Reichmann, et al., 1992; Frisch, 1994). Moreover, exogenous polypeptide factors such as Scatter Factor (SF)/Hepatocyte growth factor (HGF) and New-Regulin/HER-Regulin play important roles in the changes in the migration and differentiation properties of epithelial cells (Birchmeier et al., 1993; Hartmann et al., 1994; Soriano et al., 1995). only recently, Transforming Growth Factor 1 (TGF&bgr;1) was identified as another potent modulator of the phenotype of breast epithelial cells (Miettinen et al., 1994; Zambruno et al., 1995).
TGF&bgr;1 belongs to a large super-family of multifunctional polypeptide factors. The TGF&bgr; family itself consists of three genes, TGF&bgr;1, TGF&bgr;2 and TGF&bgr;3, which have extremely high homology with one another. In mammals the TGF&bgr;-super-family also includes the various TGF&bgr; genes as well as the embryonic morphogenes, such as e.g. the family of the activins, “Müllerian Inhibitory Substance”, and the bmp family (“Bone Morphogenetic Protein”), which play important roles both in regulating embryo development and in the reorganisation of epithelia (Roberts and Sporn, 1992). TGF&bgr;1 inhibits the growth of many cell types, including epithelial cells, but stimulates the proliferation of various types of mesenchymal cells. In addition, TGF&bgr;s induce the synthesis of extracellular matrix proteins, modulate the expression of matrix proteinases and proteinase inhibitors and change the expression of integrins. Moreover, TGF&bgr;s are expressed in large amounts in many tumours (Derynck et al., 1985; Keski-Oja et al., 1987). This strong occurrence in neoplastic tissues could indicate that TGF&bgr;s are strategic growth/morphogenesis factors which influence the malignant properties associated with the various stages of the metastatic cascade. TGF&bgr;s inhibit the growth of normal epithelial and relatively differentiated carcinoma cells, whereas undifferentiated tumour cells which lack many epithelial properties are generally resistant to growth inhibition by TGF&bgr;s (Hoosein et al., 1989; Murthy et al., 1989). Furthermore TGF&bgr;1 may potentiate the invasive and metastatic potential of a breast adenoma cell line (Welch et al., 1990), which indicates the role of TGF&bgr;1 in the tumour progression. The molecular mechanisms underlying the effect of TGF&bgr;s during the tumour cell invasion and metastasisation do, however, require further explanation.
The formation of breast cancer (mammary carcinoma) in humans involves the overexpression of (mutated or, more often, non-mutated) ras-genes and the overexpression of receptor-tyrosinekinases, which activate the Ras-signal transmission pathway (De Bortoli et al., 1985; Kern et al., 1990; LeJeune et al., 1993).
SUMMARY OF THE INVENTION
The aim of the present invention was to provide new pharmaceutical compositions for tumour therapy.
The solution to the problem started from the following findings obtained from the tests carried out:
1. The activity of TGF&bgr; on the tumour cell, in cooperation with (i) the expression of oncogenic Ras, with (ii) the overexpression of normal Ras or of receptor tyrosinekinases which activate the Ras signal transmission pathway or with (iii) other oncogenes activated in the tumour cell, lead to a conversion of epithelial cells into fibroblastoid cells with invasive potential.
2. The autocrine production of TGF&bgr; by the converted cells leads to the maintenance of the degenerate, invasive cell status.
3. Interruption of the transmission mediated by the TGF&bgr;-receptor signal prevents “epithelial-fibroblastoid conversion” (EFC) and the concomitant invasiveness and may change cells which have already undergone an EFC and are growing in a stably invasive manner back into epitheloid cells which are no longer growing invasively (fibroblastoid-epithelial conversion; FEC).
Within the scope of the present invention, the role of TGF&bgr;1 in the normal development of the mammary glands was investigated with a view to assessing possible side effects of TGF&bgr;1 inhibitors.
Within the scope of the present invention, it was shown, on the one hand, that Ha-Ras-transformed breast epithelial cells (EpRas-cells) undergo a transition (conversion) from the epithelial to the fibroblastoid (or mesenchymal) state in the formation of tumours in mice. This transition is hereinafter referred to as EF-transition or EF-conversion (“Epithelial-Fibroblastoid Cell Conversion”, EFC). Such an EF-conversion has also been demonstrated in vitro. For this, EpRas cells were cultivated in type I collagen gels. In the absence of serum these cells developed into three-dimensional, cystic hollow structures, the walls of which consisted of a single-thickness layer (monolayer) of polarised epithelial cells. TGF&bgr;1 caused these same Ras-transformed cells to develop into disorganised strands consisting of spindle-shaped cells with fibroblastoid properties. In non-transformed epithelian cells TGF&bgr;1 was unable to cause such changes. The converted cells were highly invasive both in collagen gels and in chicken heart invasion assays. Surprisingly it was found that, once the fibroblastoid cells had undergone the conversion, they themselves produced large amounts of TGF&bgr;1. If this self-produced TGF&bgr;1 was inactivated by a TGF&bgr;1 neutralising antibody, the cells changed back into a polarised, epithelial phenotype. This cell behaviour indicates that the converted fibroblastoid phenotype is maintained by TGF&bgr;1, the TGF&bgr;1 acting through an autocrine loop.
It was also shown, within the scope of the present invention, that the mechanism observed in vitro also applies in vivo: tumour cells which had undergone an EF-conversion themselves produced TGF&bgr;1. Moreover, TGF&bgr;1 is capable of triggering and sustaining the invasive phenotype of Ha-Ras-transformed breast epithelial cells in experimentally induced tumours.
Moreover, it was shown within the scope of the present invention that in human tumours of various origins
Beug Hartmut
Heider Karl-Heinz
Oft Martin
Reichmann Ernst
Boehringer Ingelheim International GmbH
Caputa Anthony C.
Harris Alana M.
Sterne Kessler Goldstein & Fox P.L.L.C.
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