In vivo molecular imaging of met tyrosine kinase growth factor receptor activity in normal organs and breast tumors.

Molecular imaging techniques allow visualization of specific gene products and their physiological processes in living tissues. In this study, we present a new approach for molecular imaging of endogenous tyrosine kinase receptor activity. Met and its ligand hepatocyte growth factor scatter factor (HGF/SF), which mediate mitogenicity, tumorigenicity, and angiogenesis, were used as a model. HGF/SF and Met play a significant role in the pathogenesis and biology of a wide variety of human epithelial cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have shown previously that in vitro activation of Met by HGF/SF increases oxygen consumption. In this study, we demonstrate that Met activation in vivo by HGF/SF alters the hemodynamics of normal and malignant Met-expressing tissues. Tumor-bearing BALB/C mice were i.v. injected with HGF/SF and imaged using magnetic resonance imaging (MRI) and Doppler ultrasound. Organs and tumors expressing high levels of Met showed the most substantial alteration in blood oxygenation levels as measured by blood oxygenation level depended (BOLD)-MRI. No significant alteration was observed in tumors or organs that does not express Met. In the liver, which expresses high levels of Met, MRI signal alteration of about 60% was observed. In the kidneys, signal alteration was approximately 30%, and no change was observed in muscles. The extent of MRI signal alteration was also in correlation with HGF/SF doses. Injection of 7 and 170 ng/g body weight resulted in signal alteration of 5% and 30%, respectively, in tumors. Doppler ultrasound measurements demonstrated that these MRI changes are at least partially attributable to altered blood flow. These hemodynamic alterations, measured by MRI and Doppler ultrasound, were used in this study for the molecular imaging of Met activity in vivo. This novel molecular imaging technique may be used for in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.

Dominant negative Met reduces tumorigenicity-metastasis and increases tubule formation in mammary cells.

Activation of the Met tyrosine kinase growth factor receptor by its ligand HGF/SF has been shown to increase in vitro invasiveness in epithelial cell lines. To study the effect of Met-HGF/SF signaling in breast cancer cells, we transfected met, hgf/sf and dominant negative (DN) forms of met into the poorly differentiated metastatic murine mammary adenocarcinoma cell line DA3. These cells express moderate levels of endogenous Met, which is rapidly phosphorylated in response to HGF/SF treatment. Met+hgf/sf transfection results in significantly increased tumorigenic and metastatic activity in vivo accompanied by reduced tubule formation. DA3 cells transfected with DN forms of Met (DN-DA3) exhibit reduced Met phosphorylation following exposure to HGF/SF. Furthermore, as compared to the parental cells, the DN-DA3 cells exhibit diminished in vitro scattering and invasiveness, while in vivo they display greatly reduced tumorigenicity and spontaneous metastasis. Tumors emanating from DN-DA3 cells injected to BALB/C mice are highly differentiated and display extensive tubule formation. These results suggest that Met-HGF/SF signaling is a determining factor in the delicate balance between differentiation/tubule formation and tumorigenicity-metastasis. Oncogene (2000) 19, 2386 - 2397

Alteration of Met protooncogene product expression and prognosis in breast carcinomas.

OBJECTIVE: To objectively quantify the expression and prognostic implications of the met protooncogene product (Met) in human breast cancer. STUDY DESIGN: One hundred eighty-two cases of primary human breast cancer were collected. Both the normal and tumor portions of the original surgical pathology specimen were immunostained for Met and imaged using laser scanning confocal microscopy. Then the cases were ranked according to relative concentrations of normal and tumor Met expression. Subsequently, they were quantified using image analysis and the results correlated with clinical outcome to determine the prognostic value of relative levels of Met. RESULTS: Using a quantitative index to evaluate the relative levels of Met expression, high levels of Met expression in the tumor as compared with the adjacent normal ducts predicted poor prognosis for overall survival and metastasis-free survival. The risk ratio for elevated Met expression was 3.94 (P = .0009). This new method also allows determination of the clinical relevance of low levels of Met in the tumor. The overall survival between the patient population with higher, lower and unchanged levels of Met in normal tissue as compared to tumor were significantly different (P = .0020). CONCLUSION: Our studies suggest that in a subpopulation of node-negative breast cancer patients, either high or low levels of Met in tumor tissue relative to normal tissue is an indicator of poor overall survival (P = .0068). Thus, Met expression could be useful for identifying node-negative patients who could benefit from adjuvant therapy.

Isolation and characterization of an expressed hypervariable gene coding for a breast-cancer-associated antigen.

A human gene and cDNA coding for a breast-cancer-associated antigen (H23Ag) were isolated and characterized. The gene contains two exons and one intron. Part of the second exon is a tandem repeat array (TRA) consisting of multiple 60-bp G + C-rich units. We report here the characterization of unique sequences that are found in the H23Ag gene and cDNA, in addition to the 60-bp repeats. Analysis of the cDNA sequences revealed a putative ATG start codon preceded by two overlapping initiation consensus sequences (CCACC). The open reading frame determines an amino acid (aa) sequence consisting of three regions. The first region contains an initiating methionine and a highly hydrophobic putative signal peptide. This is followed by a variable number of highly conserved 20-aa repeat units (TRA). The last region, C-terminal to TRA, contains four potential N-linked glycosylation sites. The genomic nucleotide sequences demonstrate a putative promoter region that includes a 'TATA' box. A putative estrogen regulatory element is located 5' to the promoter region. The characterization of the gene and cDNA coding for the H23Ag presented here, may help to elucidate its possible function in human breast cancer.

A transcribed gene, containing a variable number of tandem repeats, codes for a human epithelial tumor antigen. cDNA cloning, expression of the transfected gene and over-expression in breast cancer tissue.

A monoclonal antibody, H23, that specifically recognizes a breast-tumor-associated antigen, was used to isolate a cDNA insert that codes for the antigenic epitope. Nucleotide sequencing of this cDNA, as well as a longer 850-bp cDNA insert, shows that they are composed of 60-bp (G + C)-rich tandem repeating units. The coding strand was determined and codes for a proline-rich 20-amino-acid repeat motif. A comparison of the highly conserved repeat unit with the deduced flanking amino acid sequences demonstrates conservation of specific subregions of the repeat consensus within the flanking amino acids. Hybridization of the 60-bp cDNA probe with RNAs extracted from a variety of primary and metastatic human tumors yields relatively high levels of hybrid with the breast carcinomas, as compared to lower hybrid levels with RNAs from other epithelial tumors. RNA extracted from breast tissue adjacent to the tumor or from benign breast tumors, demonstrates low or undetectable levels of hybridization. Probing Southern blots with the 60-bp repeat shows that the tumor antigen is highly polymorphic and contains a variable number of tandem repeats (VNTRs). The VNTR nature of the gene was confirmed by probing Southern blots with unique genomic sequences that are physically linked to an isolated gene fragment that also contains the tandem repeat array. Mouse cells transfected with this gene fragment produce tumor antigen that is readily detected by H23 monoclonal antibodies. The allelic forms seen in 10 different primary human tumors demonstrate 100% concordance with the various mRNA species expressed. These studies are extended to the protein forms detected by immunoblot analyses that show both a correlation of the expressed tumor antigen species with the allelic forms as well as significantly increased expression in breast cancer tissue. The above studies unequivocally establish the over-expression of a VNTR gene coding for an epithelial tumor antigen in human breast cancer tissue.

Human epithelial tumor antigen cDNA sequences. Differential splicing may generate multiple protein forms.

The isolation and characterization of complementary DNAs (cDNAs) which code for an epithelial antigen aberrantly expressed in human breast tumor tissue are described here. The only information regarding the primary structure of this potentially important antigen has been a 20-amino-acid repeat motif. We now report the complete amino acid sequences of different forms of the human epithelial tumor antigen as deduced from the nucleotide sequence of isolated non-repeat cDNAs. The diversity of protein forms is generated by a series of alternative splicing events that occur in the regions located upstream and downstream to a central tandem repeat array. Isolated cDNAs coding for the upstream region show that differential usage of alternative splice acceptor sites may generate two protein forms containing putative signal peptides of varying hydrophobicities. The complexity of possible antigen forms is further compounded by alternative splicing events occurring in the region 3' to the repeat array. The isolated cDNAs 3' to the tandem repeats indicate that whereas one mRNA transcript is colinear with the gene, and defines an open reading frame (ORF) containing 160 amino acids downstream to the repeat array, a second cDNA correlates with a mRNA that is generated by a series of splicing events. The deduced amino acid sequence of the spliced cDNA contains an ORF that is identical for 149 amino acids downstream to the repeat array with the amino acid sequence of the unspliced cDNA. At this point it diverges and continues for an additional 179 amino acids. The sequence contains a highly hydrophobic 28-amino-acid peptide, located towards the carboxyl terminus, that may correspond to a transmembrane region. The cDNAs and deduced amino acid sequences, presented here, define the complete amino acid sequences of the epithelial tumor antigen and demonstrate the existence of multiple protein forms that probably localize to different cellular and extracellular compartments.