Takemi Otsuki

Expression of vascular endothelial growth factor (VEGF) family members in breast cancer

Vascular endothelial growth factor (VEGF)‐A is known to play an important role in tumor angiogenesis. Three additional members of the VEGF family, VEGF‐B, ‐C and ‐D, have recently been discovered. VEGF‐C and VEGF‐D are ligands for VEGF receptor‐3, which is expressed in the endothelium of lymphatic vessels. The expression of VEGF‐C is known to be associated with the development of lymphatic vessels. Therefore, it is conceivable that VEGF‐C and VEGF‐D might play a role in the development of lymphatic vessels in solid tumors. To obtain some clue as to this role, we developed a semi‐quantitative reverse transcription‐polymerase chain reaction method to investigate the mRNA expression levels of each VEGF family member in breast cancer. All the VEGF family members were expressed at different levels in seven human breast cancer cell lines explored. Although VEGF‐A and VEGF‐B expressions were detected in both node‐positive and node‐negative breast tumors, VEGF‐C expression was detected only in node‐positive tumors. VEGF‐D expression was detected only in an inflammatory breast cancer and a tumor which developed an inflammatory skin metastasis. These findings suggest a possible relationship between the expression level of VEGF‐C and/or VEGF‐D and the development of lymphatic tumor spread.

Targeting LDH enzymes with a stiripentol analog to treat epilepsy

Targeting metabolism to tackle seizures About 1% of us suffer from epilepsy. Unfortunately, presently available drugs do not work for a third of epileptic patients. Sada et al. wanted to develop compounds to treat drug-resistant epilepsy (see the Perspective by Scharfman). They focused on a metabolic pathway in the brain, the astrocyte-neuron lactate shuttle. They found that lactate dehydrogenase, a key molecule in nerve cell metabolism, controls brain excitability. Searching for a substance that selectively targets this molecule, they found a potential anti-epileptic drug that strongly suppressed drug-resistant epilepsy in an animal model. Science, this issue p. 1362; see also p. 1312 A new drug that acts on lactate dehydrogenase causes neuronal hyperpolarization and decreases spiking activity in a mouse model of epilepsy. [Also see Perspective by Scharfman] Neuronal excitation is regulated by energy metabolism, and drug-resistant epilepsy can be suppressed by special diets. Here, we report that seizures and epileptiform activity are reduced by inhibition of the metabolic pathway via lactate dehydrogenase (LDH), a component of the astrocyte-neuron lactate shuttle. Inhibition of the enzyme LDH hyperpolarized neurons, which was reversed by the downstream metabolite pyruvate. LDH inhibition also suppressed seizures in vivo in a mouse model of epilepsy. We further found that stiripentol, a clinically used antiepileptic drug, is an LDH inhibitor. By modifying its chemical structure, we identified a previously unknown LDH inhibitor, which potently suppressed seizures in vivo. We conclude that LDH inhibitors are a promising new group of antiepileptic drugs.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

Anti-HER2 antibody enhances the growth inhibitory effect of anti-oestrogen on breast cancer cells expressing both oestrogen receptors and HER2

Anti-oestrogen is effective for the treatment of oestrogen receptor (ER)-positive breast carcinomas, butmost of these tumours become resistant to anti-oestrogen. It has been suggested that anti-oestrogen therapy may induce a HER2signalling pathway in breast cancer cells and this may cause resistance to anti-oestrogen. Thus, it is conceivable that combinedtherapy with anti-oestrogen and anti-HER2 antibody might be more effective. In the present study, we investigated the effect ofcombined treatment with a humanized anti-HER2 monoclonal antibody, rhumAbHER2 (trastuzumab), and an anti-oestrogen, ICI 182,780, onthe cell growth of three human breast cancer cell lines which respectively express different levels of ER and HER2. The combinedtreatment enhanced the growth inhibitory effect on ML-20 cells, which express a high level of ER and a moderate level of HER2, butshowed no additive effect on either KPL-4 cells, which express no ER and a moderate level of HER2, or MDA-MB-231 cells, which express no ER and a low level of HER2. It is also suggested that both the antibody and anti-oestrogen induce a G1–S blockade and apoptosis. These findings indicate that combined treatment with anti-HER2 antibody and anti-oestrogen may be useful for thetreatment of patients with breast cancer expressing both ER and HER2.

Overexpression of PDZK1 within the 1q12-q22 amplicon is likely to be associated with drug-resistance phenotype in multiple myeloma.

We investigated DNA copy number aberrations in 37 cell lines derived from multiple myelomas (MMs) using comparative genomic hybridization, and 11 (29.7%) showed high-level gain indicative of gene amplification at 1q12-q22. A corresponding transcriptional mapping using oligonucleotide arrays extracted three up-regulated genes (IRTA2, PDZK1, and S100A6) within the smallest region of overlapping in amplifications. Among them PDZK1 showed amplification and consequent overexpression in the MM cell lines. Amplification of PDZK1 was observed in primary cases of MM as well. MM cell lines with amplification of PDZK1 exhibited the resistance to melphalan-, cis-platin-, and vincristin-induced cell death compared with MM cell lines without its amplifications. Furthermore, down-regulation of PDZK1 with an anti-sense oligonucleotide sensitized a cell line KMS-11 to melphalan, cis-platin, and vincristin. Taken together, our results indicate that PDZK1 is likely to be one of targets for 1q12-q22 amplification in MM and may be associated with the malignant phenotype, including drug resistance, in this type of tumor.

Hypoxia reduces hormone responsiveness of human breast cancer cells

Resistance to hormonal therapy frequently occurs following successful treatment in breast cancer. The mechanism responsible for this acquired resistance is still unknown. It has been suggested that a hypoxic tumor microenvironment promotes malignant progression of cancer, i.e., hypoxia may promote estrogen‐independent growth (a more malignant phenotype) of breast cancer. To clarify this hypothesis, the effects of hypoxia on the growth responses to hormonal agents and the expression levels of estrogen receptor (ER)‐α and progesterone receptor (PgR) were investigated in two human breast cancer cell lines, ML‐20 and KPL‐1. The expression level of ER‐α was significantly decreased by hypoxia (1% O2) in a tune‐dependent manner in both cell lines. Hypoxia also significantly reduced the growth‐promoting effect of estradiol (E2) and the growth‐inhibitory effects of an antiestrogen, ICI 182 780, and a progestin, medroxyprogesterone acetate, in both cell lines. In addition, hypoxia markedly suppressed the induction of PgR mRNA and protein by E2 in both cell lines. To clarify further the effect of hypoxia on ER‐α expression, the expression levels of hypoxia‐inducible factor‐la (HIF‐lα), a marker of hypoxia and ER‐α were immunohistochemically examined in 36 breast cancer specimens. ER‐α expression (both its proportion and intensity) was significantly lower in nuclear HIF‐lα‐positive tumors than in negative tumors. These findings indicate that hypoxia down‐regulates ER‐α expression as well as ER‐α function in breast cancer cells. These processes may lead to an acquired resistance to hormonal therapy in breast cancer

A Radicicol Derivative, KF58333, Inhibits Expression of Hypoxia-inducible Factor-1α and Vascular Endothelial Growth Factor, Angiogenesis and Growth of Human Breast Cancer Xenografts

A novel oxime derivative of radicicol, KF58333, binds to the heat shock protein 90 (Hsp90) and destabilizes its associated signaling molecules. These effects play a critical role in the growth inhibition of tumor cells. To further investigate the effects of this agent, it was administered to two human breast cancer cell lines, KPL‐1 and KPL‐4, both in vitro and in vivo. KF58333 dose‐dependently inhibited the growth and vascular endothelial growth factor (VEGF) secretion, concomitantly with a decrease in VEGF mRNA expression, in each cell line. This agent also suppressed the increase of VEGF secretion and expression induced by hypoxia (1% O2). Intravenous injections of this agent into nude mice bearing either KPL‐1 or KPL‐4 xenografts significantly inhibited the tumor growth associated with a decrease in the Ki67 labeling index and microvascular area and an increase in apoptosis and the necrotic area. These findings indicate that the antitumor activity of this radicicol derivative may be partly mediated by decreasing VEGF secretion from tumor cells and inhibiting tumor angiogenesis. To explore the action mechanisms of the anti‐angiogenic effect, the expression level of hypoxia‐inducible factor (HIF)‐lα was investigated. KF58333 provided a significant decrease in the HTF‐lα protein expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of HIF‐lα was not decreased by this agent. It is suggested that the post‐transcriptional down‐regulation of HIF‐lα expression by this agent may result in a decrease of VEGF expression and tumor angiogenesis.

Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells

A high expression level of epidermal growth factor receptor (EGFR)/HER1 has been suggested to lead to a shorter survival time and resistance to endocrine therapy in patients with breast cancer. To test the hypothesis that inhibition of the EGFR signalling pathway affects the antitumour effect of endocrine therapy, an EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, and an oestrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. A total of five human breast cancer cell lines were used. The effects of single or combined treatments with gefitinib and/or fulvestrant on cell growth, cell cycle progression and apoptosis were analysed. Changes in the expression levels of cyclin-dependent kinase inhibitors, p21 and p27, an antiapoptotic factor, Bcl-2, and a proapoptotic factor, Bax, were also investigated. All cell lines tested were sensitive to gefitinib (50% growth inhibitory concentration, 10–28.5 μM). Breast cancer cell lines with a high expression level of HER1 or HER2 were more sensitive to gefitinib than the others. Gefitinib induced a significant G1–S blockade in ER-positive KPL-3C cells. Gefitinib induced significant apoptosis in HER1-overexpressing MDA-MB-231 cells. Gefitinib additively increased the antitumour effect of fulvestrant in all three ER-positive cell lines in a medium supplemented with 17β-oestradiol. The combined treatment promoted cell cycle retardation in KPL-3C cells, which is associated with an upregulation of p21 by fulvestrant and gefitinib, respectively. Apoptosis was associated with downregulation of Bcl-2 by gefitinib in MDA-MB-231 cells. These results suggest an additive interaction between the EGFR-TKI gefitinib and the antioestrogen fulvestrant in ER-positive breast cancer cells.

Over‐expression of the decoy receptor 3 (DcR3) gene in peripheral blood mononuclear cells (PBMC) derived from silicosis patients

Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, is considered to be involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). Recently, a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds FasL and inhibits FasL‐induced apoptosis, has been identified. Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities. We have found that serum soluble Fas (sFas) levels are elevated in silicosis patients and that sFas message is dominantly expressed in PBMC derived from these patients. This study examined DcR3 gene expression in PBMC derived from patients with silicosis, SLE, or progressive systemic sclerosis (PSS), and compared it with that in healthy volunteers (HV). The relative expression level of the DcR3 gene was examined in PBMC derived from 37 patients with silicosis without clinical symptoms of autoimmune disease, nine patients with SLE, 12 patients with PSS, and 28 HV using the semiquantitative multiplex‐reverse transcriptase‐polymerase chain reaction (MP‐RT‐PCR). The correlation between the relative expression level of the DcR3 gene and multiple clinical parameters for respiratory disorders and immunological abnormalities in individuals with silicosis was analysed. The DcR3 gene was significantly over‐expressed in cases of silicosis or SLE when compared with HV. In addition, the DcR3 relative expression level was positively correlated with the serum sFas level in silicosis patients. It is unclear, however, whether over‐expression of the DcR3 gene in silicosis is caused by chronic silica exposure, merely accompanies the alteration in Fas‐related molecules, or precedes the clinical onset of autoimmune abnormalities. It will be necessary to study these patients further, establish an in vitro model of human T cells exposed recurrently to silica compounds, and resolve whether the increase in DcR3 mRNA expression is a cause or consequence of disease.

SOLUBLE FAS MRNA IS DOMINANTLY EXPRESSED IN CASES WITH SILICOSIS

Although it is well known that cases with silicosis exhibit various immunological abnormalities, the mechanisms involved in the occurrence of immuno‐dysfunction or dysregulation induced by silica compounds have not yet been determined. Fas is a well‐known cell surface molecule that is involved in the apoptosis pathway that belongs to the tumour necrosis factor‐receptor family. Soluble Fas (sFas) is produced as an alternatively spliced product of the Fas gene and protects cells from apoptosis due to antagonization of the binding between membrane form of the Fas gene (mFas) and the Fas ligand. To determine the role of the Fas/Fas ligand system in silica‐induced immunological abnormalities, we investigated Fas and Fas‐ligand message expression levels using the multiplex reverse transcription–polymerase chain reaction (RT–PCR) method with peripheral blood mononuclear cells from silicosis cases with no clinical symptoms of autoimmune diseases. Although the relative expression levels of the Fas or Fas‐ligand genes were not remarkably altered in these cases, we observed the sFas message was dominantly expressed compared with mFas expression. These results suggest that self‐recognizing clones in cases with silicosis survive for decades, escaping the exclusion mechanisms induced by apoptosis. Then they cause the appearance of autoantibodies and the acquisition of autoimmune diseases sequentially.