TOP2A gene copy gain predicts response of epithelial ovarian cancers to pegylated liposomal doxorubicin: TOP2A as marker of response to PLD in ovarian cancer.

OBJECTIVE: The treatment of platinum resistant/refractory epithelial ovarian cancer (EOC) is a challenge for oncologists. One of the most utilized drugs in these patients is pegylated liposomal doxorubicin (PLD). As PLD is active only in a small subset of patients and causes side effects, selection of responsive patients is an unmet need and might be guided by the status of the DNA topoisomerase II alpha (TOP2A) that is poisoned by the drug. METHODS: From 176 ovarian cancers treated in three institutions, we selected 38 patients treated with PLD monotherapy as second/third line of treatment. TOP2A gene copies were measured using Fluorescent In Situ Hybridization (FISH) and expression evaluated using immunohistochemistry. Patients' derived xenografts (PDXs) of ovarian cancers were used to assess the correlation between TOP2A protein expression and response to PLD. RESULTS: Clinical data showed that TOP2A gene gain that is paralleled by increased expression of the protein, was associated with a higher probability of clinical benefit from PLD. Treatment of PDXs demonstrated that only xenografts showing a high percentage of TOP2A expressing cells underwent tumor shrinkage when treated with PLD. CONCLUSIONS: These data show that TOP2A gene gain and protein over-expression might predict activity of PLD in platinum resistant/refractory EOC.

Preclinical immunotherapy with Cytokine-Induced Killer lymphocytes against epithelial ovarian cancer.

Despite improvements in surgery and medical treatments, epithelial ovarian cancer (EOC) remains the most lethal gynaecological malignancy. Aim of this study is to investigate the preclinical immunotherapy activity of cytokine-induced killer lymphocytes (CIK) against epithelial ovarian cancers, focusing on platinum-resistant settings. We generated CIK ex vivo starting from human peripheral blood samples (PBMCs) collected from EOC patients. Their antitumor activity was tested in vitro and in vivo against platinum-resistant patient-derived ovarian cancer cells (pdOVCs) and a Patient Derived Xenograft (PDX), respectively. CIK were efficiently generated (48 fold median ex vivo expansion) from EOC patients; pdOVCs lines (n = 9) were successfully generated from metastatic ascites; the expression of CIK target molecules by pdOVC confirmed pre and post treatment in vitro with carboplatin. The results indicate that patient-derived CIK effectively killed autologous pdOVCs in vitro. Such intense activity was maintained against a subset of pdOVC that survived in vitro treatment with carboplatin. Moreover, CIK antitumor activity and tumor homing was confirmed in vivo within an EOC PDX model. Our preliminary data suggest that CIK are active in platinum resistant ovarian cancer models and should be therefore further investigated as a new therapeutic option in this extremely challenging setting.

Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

A positive feedback loop between hepatocyte growth factor receptor and beta-catenin sustains colorectal cancer cell invasive growth.

Overexpressed or activated hepatocyte growth factor receptor, encoded by the MET proto-oncogene, was found in the majority of colorectal carcinomas (CRCs), whose stepwise progression to malignancy requires transcriptional activation of beta-catenin. We here demonstrate that a functional crosstalk between Met and beta-catenin signaling sustains and increases CRC cell invasive properties. Hepatocyte growth factor (HGF) stimulation prompts beta-catenin tyrosine phosphorylation and dissociation from Met, and upregulates beta-catenin expression via the phosphatidylinositol 3-kinase pathway in conditions that mimic those found by the invading and metastasizing cells. Additionally, a transcriptionally active form of beta-catenin, known to be oncogenic, enhances Met expression. Furthermore, HGF treatment increases the activity of the beta-catenin-regulated T-cell factor transcription factor in cells expressing the wild-type or the oncogenic beta-catenin. In the mirror experiments, either Met or beta-catenin knocking down also reduces their protein level. In biological assays, beta-catenin knocking down abrogates the HGF-induced motile phenotype, whereas active beta-catenin fosters ligand-independent cell scattering. Met and beta-catenin also cooperate in promoting entry into the cell cycle and in protecting cells from apoptosis. In conclusion, Met and beta-catenin pathways are mutually activated in CRC cells. This might generate a self-amplifying positive feedback loop resulting in the upregulation of the invasive growth properties of CRC cells.

p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line.

A protein with an Mr of 145,000 (p145) was detected by antibodies to phosphotyrosine by Western blot (immunoblot) analysis. This protein was phosphorylated on tyrosine in a gastric carcinoma cell line. In cells that were metabolically labeled with 32Pi, this protein was phosphorylated on tyrosine and serine. p145 is a cysteine-rich transmembrane glycoprotein. The extracellular domain could be labeled by 125I under nonpermeating conditions and was cleaved by mild trypsin treatment of intact cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed a shift of p145 mobility to an apparent Mr of 190,000. After immunoprecipitation with phosphotyrosine antibodies, p145 displayed a strong associated protein kinase activity in vitro, becoming phosphorylated on tyrosine. There was no immunological cross-reaction between p145 and known tyrosine kinases. Both in vivo and in vitro tyrosine phosphorylations were unaffected by the addition of known growth factors. However, p145 was rapidly dephosphorylated in vivo when cells were exposed to low pH, a condition that is known to dissociate ligands from their receptors. These data suggest that p145 is associated with a protein tyrosine kinase activity which, in the tumor cell line studied, is activated by an as yet unidentified factor.

Expression of the Met/hepatocyte growth factor receptor in human pancreatic cancer.

The c-MET oncogene encodes the receptor for hepatocyte growth factor (HGF) scatter factor, a multifunctional cytokine able to mediate morphogenesis as well as invasive growth of epithelial cells. The c-MET-encoded receptor is detectable only at low levels in the normal human exocrine pancreas, but it is up-regulated in the majority of pancreatic ductal adenocarcinomas. The c-MET-encoded HGF receptor is also overexpressed in a proportion of the panel of 31 human pancreatic cancer cell lines examined, which have a range of different growth properties and degrees of differentiation. In most cases the HGF receptor found in the malignant cells has features of the normal receptor. When added to pancreatic cancer cell lines, HGF triggers receptor phosphorylation and stimulates cells to move and proliferate. In overexpressing cell lines, the Met/HGF receptor is phosphorylated in the absence of endogenously produced or exogenously added ligand. These data suggest that the Met/HGF receptor may be involved in the growth and behavior of pancreatic cancer and may contribute to the ductal phenotype of these tumors.

Biosynthesis of the protein encoded by the c-met proto-oncogene.

The proto-oncogene c-met encodes a transmembrane protein with structural features of a growth factor receptor. We have previously shown that the c-met protein (c-Met) is a heterodimer of two disulphide linked chains of 50 kd (alpha) and 145 kd (beta). In this work we have studied the biosynthesis of the c-met product in a gastric carcinoma cell line (GTL-16) where the c-met gene is amplified and overexpressed. Following metabolic labelling of the cells in the presence of tunicamycin, anti-met antibodies immunoprecipitate a protein of 150 kd. In pulse-chase experiments carried out in the absence of tunicamycin, a 170 kd product appears first. Within the next few minutes, this precursor modifies its SDS migration, probably as a consequence of modification(s) of its intra-chain disulphide bonds. After 45 min of chase, this single polypeptide precursor is cleaved to form a 50 kd alpha subunit and a 145 kd beta subunit that are joined by disulphide bonds in an alpha beta complex with an apparent molecular weight of 190 kd. The presence of N-linked oligosaccharides in both the precursor and the mature protein was shown by enzymatic de-glycosylation of the immunoprecipitated proteins. The half-life of the mature protein was calculated to be approximately 5h. The c-met protein has similar structure and biosynthesis in other human cell lines.

Protein kinase-c activation inhibits tyrosine phosphorylation of the c-met protein.

The mature product of the c-met proto-oncogene is a putative tyrosine kinase receptor of 190 kd with an alpha beta heterodimeric structure. The c-met protein is phosphorylated in vivo on the beta subunit in the gastric carcinoma cell line GTL-16 (Giordano et al., 1988). Western blots with phosphotyrosine antibodies show that tyrosine phosphorylation of the beta subunit is reduced by treatment of GTL-16 cells with protein kinase C activators (tumor promoting phorbol esters such as phorbol 12-myristate 13-acetate, TPA, and beta-phorbol 12,13-dibutyrate, PdBu, or membrane permeable synthetic diacylglycerol 1-oleyl-2-acetyl-sn-glycerol, OAG). The inactive analog alpha-phorbol 12,13-didecanoate has no effect. The inhibition induced by TPA is dose dependent and maximal after 1 h. Depletion of protein kinase-C by prolonged treatment with TPA (18-48 h) increases the phosphorylation on tyrosine of the beta subunit. Phospho-amino acid analysis of the c-met protein immunoprecipitated from [32P]orthophosphate-labelled GTL-16 cells shows that protein kinase-C activation leads to an increase in serine phosphorylation and to concomitant decrease in tyrosine phosphorylation. These results suggest that, similar to the EGF and insulin receptor, the putative receptor encoded by the c-met proto-oncogene may be negatively modulated by protein kinase-C phosphorylation.

The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit.

The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.

Expression of the Met/HGF receptor in normal and neoplastic human tissues.

The MET oncogene encodes a transmembrane tyrosine kinase receptor. Recently, hepatocyte growth factor (HGF), a potent growth factor for hepatocytes involved in liver regeneration, has been proposed as a ligand. In this paper, the physiological role of the human Met/HGF receptor is investigated by studying its specific distribution in normal and neoplastic tissues. Northern blot analysis has shown that the MET gene is selectively expressed in several epithelial tissues. High levels of MET mRNA have been found in liver, gastrointestinal tract, thyroid and kidney. Western blot analysis has shown that the levels of the Met protein generally correspond to those of the mRNA. However, in the thyroid, where there is a high level of MET mRNA, the protein was barely detectable, suggesting translational or post-translational regulation. The protein was also detected in the brain. Normal or increased levels of MET mRNA and Met protein were consistently found in fresh samples of carcinomas as well as in epithelial tumor cell lines. In thyroid carcinomas of a specific histiotype the amount of Met protein, almost undetectable in the normal counterpart, was found to be increased more than 100-fold. The tissue distribution of the Met/HGF receptor indicates that this molecule is involved in growth control of epithelial cells other than hepatocytes and suggests that its increased expression may confer a growth advantage to neoplastic cells.

Overexpression of the c-MET/HGF receptor gene in human thyroid carcinomas.

The receptor for Hepatocyte Growth Factor is a transmembrane tyrosine kinase encoded by the c-MET oncogene. We have previously shown that the Met protein is expressed in several human epithelial tissues. The receptor is barely detectable, however, in normal thyroids and in specimens from patients affected by non-neoplastic thyroid diseases. Now we report that the expression of the Met/HGF receptor is increased a hundred fold in 22 out of 41 human carcinomas derived from the thyroid follicular epithelium. A comprehensive analysis of 15 cases showed that the overexpressing carcinomas belong to histotype variants correlated with negative prognosis and in all but one case there were evidences of locally advanced disease and/or distant metastases. The 11 benign adenomas and the 5 medullary carcinomas tested were negative. Western blot analysis with monoclonal antibodies directed against either the intracellular or the extracellular receptor domains failed to reveal major structural alterations. Southern blot analysis also demonstrated that the c-MET gene was not amplified nor rearranged. These data suggest a role for the overexpression of c-MET oncogene in the pathogenesis and progression of thyroid tumors derived from the follicular epithelium.

Selective expression of the Met/HGF receptor in human central nervous system microglia.

The c-MET proto-oncogene encodes the tyrosine kinase receptor for hepatocyte growth factor (HGF), also known as scatter factor, a powerful mitogen and motility factor for epithelial cells. We now show that the two previously described forms of the Met/HGF receptor, the intact p190MET and the truncated p140MET, are expressed in physiological conditions in the human central nervous system (CNS). The receptors were identified by Western blot analysis with monoclonal antibodies directed against different epitopes. By immunohistochemical staining the Met/HGF receptor was found to be expressed in a homogeneous cell population, equally distributed between the grey and the white matter, showing morphological features and immunochemical markers specific for the resident microglial cells. These data suggest a possible role for the c-MET proto-oncogene and HGF in microglial reactions to CNS injuries.

Somatic mutations of the MET oncogene are selected during metastatic spread of human HNSC carcinomas.

A metastatic cancer develops by accumulation of mutations in genes that control growth, survival and spreading. The latter genes have not yet been identified. In lymph node metastases of head and neck squamous cell carcinomas (HNSCC), we found mutations in the MET oncogene, which encodes the tyrosine kinase receptor for Scatter Factor, a cytokine that stimulates epithelial cell motility and invasiveness during embryogenesis and tissue remodeling. We identified two somatic mutations: the Y1230C, known as a MET germline mutation which predisposes to hereditary renal cell carcinoma, and the Y1235D that is novel and changes a critical tyrosine, known to regulate MET kinase activity. The mutated MET receptors are constitutively active and confer an invasive phenotype to transfected cells. Interestingly, cells carrying the MET mutations are selected during metastatic spread: transcripts of the mutant alleles are highly represented in metastases, but barely detectable in primary tumors. These data indicate that cells expressing mutant MET undergo clonal expansion during HNSCC progression and suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis.

Retrogenic expression of the MET proto-oncogene correlates with the invasive phenotype of human rhabdomyosarcomas.

The MET oncogene encodes the receptor for HGF/Scatter Factor, known to control cell motility and invasion in epithelial cells. We report that the Met/HGF receptor, absent in differentiated adult skeletal muscles, is aberrantly expressed in clinical samples and in established cell lines of human rhadbomyosarcomas. In both the embryonal and alveolar histotypes the oncogene is overexpressed and, in some cases, amplified. The Met receptor is exposed at the cell surface and is functionally active in response to HGF/Scatter Factor. Accordingly, rhabdomyosarcoma cells exhibit an invasive phenotype in vitro in response to exogenous HGF/Scatter factor. As the factor is known to be produced by connective tissues, a paracrine stimulation of rhabdomyosarcoma invasiveness in vivo is hypothesized. Two alveolar rhabdomyosarcomas were found in co-express the ¿two-kringle' alternatively-spliced HGF/Scatter Factor variant, which has been previously shown to stimulate cell motility and matrix invasion in vitro. These cells displayed the invasive phenotype in the absence of exogenous HGF/Scatter Factor, suggesting an autocrine mechanism in vivo. These data indicate that aberrant expression of the MET proto-oncogene provides rhabdomyosarcoma cells with the same property as embryonal myoblasts to migrate into the surrounding connective tissues.

Overexpression of the RON gene in human breast carcinoma.

Constitutive activation of the RON gene, known to code for the tyrosine-kinase receptor for Macrophage Stimulating Protein (also known as Scatter Factor 2), has been shown to induce invasive-metastatic phenotype in vitro. As yet, nothing is known about the expression of this novel member of the MET-oncogene family in spontaneously occurring human cancers. Here we report that Ron is expressed at abnormally high levels in about 50% primary breast carcinomas (35/74 patients). Among these, the expression is increased more than 20-fold in 12 cases and the overexpressed protein is constitutively phosphorylated on tyrosine residues. Notably, Ron is only barely detectable in epithelial cells of the mammary gland, and its expression remains unchanged in benign breast lesions (including adenomas and papillomas). Overexpression was observed in different histotypic variants of carcinomas; it is associated with the disease at any stage and correlates with the post-menopausal status. In breast carcinoma cells grown in vitro, activation of the Ron receptor resulted in proliferation, migration and invasion through reconstituted basement membranes. Altogether, these data suggest a role for the RON gene in progression of human breast carcinomas to the invasive-metastatic phenotype.

Overexpression and amplification of the met/HGF receptor gene during the progression of colorectal cancer.

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor, a potent mitogen for epithelial cells that also promotes cell motility and invasiveness. We have studied the changes of c-met gene expression that occur during the progression of colorectal tumors. Sixteen adenomas, 123 primitive carcinomas, and 25 liver metastases were examined. In several instances it was possible to compare same-patient samples of normal colon mucosa against primary tumor and primary carcinoma against synchronous metastasis. The expression of the c-met gene was increased from 5- to 50-fold in about 50% of tumors, at any stage of progression, and in 70% of liver metastases. Overexpression was associated with amplification of the c-met gene in only 10% of carcinomas, but in 8 of 9 metastases examined. These data suggest that overexpression of the c-met oncogene contributes a selective growth advantage to neoplastic colorectal cells at any stage of tumor progression. Moreover, amplification appears to give a further selective advantage for the acquisition of metastatic potential.

Negative/low expression of the Met/hepatocyte growth factor receptor identifies papillary thyroid carcinomas with high risk of distant metastases.

To investigate the clinical impact of Met/hepatocyte growth factor receptor (HGF-R) expression in thyroid cancer we studied 163 thyroid carcinomas (129 papillary, 21 follicular, and 13 anaplastic) from patients followed-up for 25-147 months postthyroidectomy. Forty-nine thyroid adenomas were also studied. Met/HGF-R expression was evaluated by semiquantitative immunohistochemistry, measuring both the proportion (scale of 0-5) and the intensity (scale, 0-5) of stained cells and calculating a total score (scale of 0-10). Met/HGF-R was absent in the normal thyroid tissue, absent or focally expressed in follicular and anaplastic tumors, and expressed at various levels in most papillary carcinomas, including microcarcinomas. Papillary carcinomas were thus categorized as having negative/low Met/HGF-R (n = 50; total score, < or = 5) or high Met/HGF-R expression (n = 70; total score, > 5). High Met/HGF-R was inversely associated with vascular invasion (P = 0.0308), but not with other prognostic factors. Negative/low Met/HGF-R expression was the most effective predictor by multivariate Cox analysis of distant metastases (hazard ratio = 9.71; P = 0.0036), higher than extrathyroid invasion (hazard ratio = 4.25; P = 0.0181), age (< or = 45 vs. > 45 yr; hazard ratio = 3.99; P = 0.0099), and vascular invasion (hazard ratio = 3.19; P = 0.0358). These findings suggest a role for Met/HGF-R in papillary thyroid cancer and its clinical use to select patients with a high risk of distant metastases.

Role of heterochromatin variation in the instability of a marker chromosome during tumor progression.

Karyotypic evolution of the poorly metastasizing tumorigenic RSV-transformed B77-3T3 fibroblast line was investigated both in highly metastasizing clones (selected by growth in hard agar) and in spontaneous metastases. Analysis of structural chromosome aberrations associated with the transition from the nonmetastatic to the metastatic phenotype was focused on a readily identifiable marker chromosome (A), displaying an extracentromeric heterochromatic region as a main feature promoting genetic instability. Well-defined changes in the structure of this marker were observed, both in vitro and in vivo, and invariably involved C-heterochromatic variation. In the metastatic clones, a specific rearrangement of the A chromosome was selected. This structural variant (B) showed two extracentromeric C-positive regions and probably originated from duplication of the segment of A included between the centromere and the internal C-band. On the other hand, selection of a modified form of chromosome A, not displaying the interpolated C-heterochromatin, had occurred in the extremely rare B77-3T3 spontaneous metastases. The connection among heterochromatin variants, genetic instability, and chromosome aberrations is discussed.

Lipid characteristics of RSV-transformed Balb/c 3T3 cell lines with different spontaneous metastatic potentials.

To determine whether a metastatic phenotype may be correlated with a characteristic lipid pattern, we compared the lipid composition of low metastasizing Balb/c 3T3 cells transformed by the B77 strain of Rous sarcoma virus (B77-3T3 cells) with that of a subclone isolated by growth in 0.6% agar, the B77-AA6 cells, which exhibit a high capacity for spontaneous metastasis. B77-3T3 cells revealed characteristics in their lipid composition common to other systems of transformed cells, i.e., an accumulation of ether-linked lipids, a reduction of the more complex gangliosides, an increase of oleic acid (18:1) and a decrease of arachidonic (20:4) and C22 polyunsaturated fatty acids in phospholipids. High metastatic B77-AA6 cells showed: a) an even more marked decrease of complex gangliosides; b) a more pronounced increase of 18:1 and decrease of 20:4 and 22 polyunsaturated fatty acids in certain phospholipid classes; and c) a higher percentage of alkyl-acyl subfractions in both phosphatidylcholine and phosphatidylethanolamine than B77-3T3 cells. Comparing the data for other systems of metastatic cells with those of lipid studies of spontaneously metastasizing B77-AA6 cell system leads us to conclude that the metastatic phenotype is characterized by a change in ether-linked lipids, rather than in fatty acids.

High chemotactic motility and growth in hard agar of a variant of RSV-transformed fibroblasts are lost in late passages.

Cloning efficiency in hard agar (0.6%) and high chemotactic migration toward fibroblast conditioned medium have been shown to characterize metastatic tumor cells. We studied growth in 0.6% agar and chemotaxis of two lines of Rous Sarcoma virus-transformed Balb/c3T3 cells, B77/3T3 (low metastatic) and AA12 (high metastatic), and compared them to their non-transformed counterpart, in order to verify whether these properties were maintained during several subcultivations. Cells were cryopreserved at early passages and thawed for experiments. Both assays were performed on freshly thawed cells (4-6 weeks in culture) and on cells which had been cultured 15-20 weeks after thawing. B77/3T3, which are tumorigenic but low metastatic and which have a very low cloning efficiency in hard agar (0.1-1%), showed a chemotactic response to Balb/c3T3 conditioned medium about two-fold higher than control Balb/c3T3. This response did not change with time in culture. AA12 cells, a genetic unstable variant of B77/3T3 selected for its growth in hard agar (0.6%), had a high cloning efficiency in hard agar and showed a high chemotactic motility (three-fold the controls). High growth in 0.6% agar and high chemotaxis of AA12 were lost in late passages, where cells behaved as the controls. It seems that besides the already reported variation in anchorage-independent growth, genetically unstable tumor cells can also have important variations in chemotactic motility during subcultivations.