Yoshifumi Nakayama

Monocarboxylate transporters 1 and 4 are involved in the invasion activity of human lung cancer cells.

Cancer cells show constitutive upregulation of glycolysis, and the concentration of lactate thus produced correlates with prognosis. Here, we examined whether lactate concentration and lactate transporter expression are related to migration and invasion activity. We found that the expression of the monocarboxylate transporters MCT1 and MCT4, but not MCT5, in human lung cancer cell lines was significantly correlated with invasiveness. To clarify the effects of MCT1 and MCT4 expression on invasion, we performed migration and invasion assays after transfection with siRNA specific for MCT1 or MCT4. Knockdown of MCT1 or MCT4 did not influence cell migration but reduced invasion; this was also observed for knockdown of the lactate transporter‐associated protein basigin. We also demonstrated that both expression and activity of MMP9 and MMP2 were not correlated with invasion activity and not regulated by MCT1, MCT4 and basigin. Furthermore, the addition of lactate did not increase migration and invasion activity, but low concentration of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS), a general anion channel blocker, as well as other MCT inhibitors quercetin and simvastatin, inhibited cell invasion without influencing migration activity and the cellular expression of MCT1 and MCT4. This is the first report suggesting that lactate transporters are involved in human cancer cell invasiveness. As such, these proteins may be promising targets for the prevention of cancer invasion and metastasis. (Cancer Sci 2011; 102: 1007–1013)

Expression of UDP-N-acetyl-α-D-galactosamine–polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma

Tumor development usually is accompanied by alterations of O‐glycosylation. Initial glycosylation of mucin‐type, O‐linked proteins is catalyzed by one of the UDP‐GalNAc–polypeptide N‐acetyl‐galactosaminyl transferases, such as GalNAc‐T3, which is expressed in adenocarcinoma cells. The authors investigated whether such expression influenced tumor differentiation or prognosis in patients with colorectal carcinoma.

Human mitochondrial transcription factor A binds preferentially to oxidatively damaged DNA.

Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mtDNA, and is also one of the high mobility group (HMG) proteins that preferentially binds to cisplatin-damaged DNA. In this study we confirmed the preferential binding of mtTFA to cisplatin-damaged DNA, and also discovered that mtTFA binds to oxidatively damaged DNA. The affinity for oxidatively damaged DNA of mtTFA is higher for A/8-oxo-dG and C/8-oxo-dG than for G/8-oxo-dG and T/8-oxo-dG. Our findings suggest that mtTFA plays an important role in the recognition of oxidative DNA damage.

Lymphatic Microvessel Density is an Independent Prognostic Factor in Colorectal Cancer

PurposeAlthough lymph node metastasis via lymphatic vessels often is related with an adverse outcome, it is not well known whether lymphatic spread to lymph node needs the development of the new lymphatic formation. In addition, the correlation between lymphangiogenesis and prognosis has not been well documented. This study was designed to assess the prognostic value of lymphangiogenesis and lymphatic vessel invasion in colorectal cancer.MethodsWe examined 106 colorectal cancer specimens by immunostaining for podoplanin, lymphatic endothelial specific marker. We evaluated lymphangiogenesis, as measured by lymphatic microvessel density, and lymphatic vessel invasion. We next investigated the association of these two parameters with the clinicopathologic findings and prognosis.ResultsA significant correlation was observed between high lymphatic microvessel density and positive lymphatic vessel invasion (P = 0.0003). Positive lymphatic vessel invasion was significantly associated with the presence of lymph node metastasis (P = 0.0071). The survival curves demonstrated that both high lymphatic microvessel density and positive lymphatic vessel invasion were correlated with an adverse outcome (P = 0.0004 and P = 0.009, respectively). In a univariate analysis, high lymphatic microvessel density and positive lymphatic vessel invasion were negatively associated with the overall survival (P = 0.0011 and P = 0.0118, respectively). Furthermore, high lymphatic microvessel density, but not lymphatic vessel invasion, correlated with a poor outcome in a multivariate analysis (P = 0.0114).ConclusionsOur data suggested that lymphatic vessel invasion was related with lymph node metastasis and that both lymphatic microvessel density and lymphatic vessel invasion were related with an adverse outcome in colorectal cancer. Furthermore, lymphatic microvessel density may be a useful prognostic factor in colorectal cancer.

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: Involvement of transforming growth factor-α

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.

The type III inositol 1,4,5-trisphosphate receptor is associated with aggressiveness of colorectal carcinoma

The inositol 1,4,5-trisphosphate receptor (InsP3R) mediates Ca(2+) signaling in epithelia and regulates cellular functions such as secretion, apoptosis and cell proliferation. Loss of one or more InsP3R isoform has been implicated in disease processes such as cholestasis. Here we examined whether gain of expression of InsP3R isoforms also may be associated with development of disease. Expression of all three InsP3R isoforms was evaluated in tissue from colorectal carcinomas surgically resected from 116 patients. Type I and II InsP3Rs were seen in both normal colorectal mucosa and colorectal cancer, while type III InsP3R was observed only in colorectal cancer. Type III InsP3R expression in the advancing margins of tumors correlated with depth of invasion, lymph node metastasis, liver metastasis, and TNM stage. Heavier expression of type III InsP3R also was associated with decreased 5-year survival. shRNA knockdown of type III InsP3R in CACO-2 colon cancer cells enhanced apoptosis, while over-expression of the receptor decreased apoptosis. Thus, type III InsP3R becomes expressed in colon cancer, and its expression level is directly related to aggressiveness of the tumor, which may reflect inhibition of apoptosis by the receptor. These findings suggest a previously unrecognized role for Ca(2+) signaling via this InsP3R isoform in colon cancer.

Prognostic significance of UDP-N-acetyl-α-D-galactosamine : polypeptide N-acetylgalactosaminyltransferase-3 (GalNAc-T3) expression in patients with gastric carcinoma

Aberrant glycosylation occurs during development of gastric carcinomas. The initiation of mucin‐type O‐glycosylation is regulated by GalNAc‐T3 (UDP‐N‐acetylgalactosamine:polypeptide N‐acetyl‐galactosaminyltransferase‐3). However, the clinical significance of GalNAc‐T3 expression in human gastric carcinoma has not yet been demonstrated. In the present study, we investigated the relationship between immunohistochemical GalNAc‐T3 expression and various clinicopathologic factors, including prognosis, in 117 gastric carcinoma patients. Of 117 gastric carcinomas examined, 59 (50.4%) showed strong expression of GalNAc‐T3. Strong expression was detected in 38 of 59 (64.4%) differentiated type and in 21 of 58 (36.2%) undifferentiated gastric carcinomas, indicating that the expression of GalNAc‐T3 correlated significantly with tumor differentiation (P=0.0023, x2 test). Overall 5‐year survival rate in patients with strong GalNAc‐T3 expression (71.0%) was significantly better than that of patients with weak expression (49.3%) (P=0.0197, log‐rank test). Multivariate analysis identified GalNAc‐T3 expression as an independent prognostic factor (P=0.0158, Cox proportional hazards model). Our data suggest that GalNAc‐T3 expression may be a useful marker for prognosis and differentiation of gastric carcinomas. (Cancer Sci 2003; 94: 32–36)

Cooperative Roles of Hepatocyte Growth Factor and Plasminogen Activator in Tubular Morphogenesis by Human Microvascular Endothelial Cells

Epidermal growth factor (EGF) or transforming growth factor‐α (TGF‐α) stimulated cell migration, chemotaxis, and the expression of tissue‐type plasminogen activator (t‐PA) in human omental microvascular endothelial (HOME) cells. Hepatocyte growth factor (HGF) stimulated cell proliferation, but had a negligible stimulatory effect on cell migration, the expression of t‐PA and tube‐like formation into collagen gel in HOME cells. Basic fibroblast growth factor stimulated cell proliferation, cell migration, tubulogenesis and the expression of urokinase‐type plasminogen activator (u‐PA) in bovine aortic endothelial (BAE) cells. HOME and BAE cells had both high‐ and low‐affinity receptors for HGF. In BAE cells, u‐PA activity and tube‐like structures in collagen gel were induced in the presence of HGF alone. In contrast, in HOME cells, t‐PA activity and tube‐like structures were induced in the presence of TGF‐a alone, but not in the presence of HGF alone. However, we observed a marked induction of tube formation by HOME cells when both t‐PA and HGF were added simultaneously. In the model system for tumor angiogenesis, when HOME cells were co‐cultured with a renal cancer cell line, KPK13, tube‐like structures were induced in the presence of HGF: KPK13 cells expressed large amounts of t‐PA mRNA. Our present study suggested that HGF in concert with active t‐PA could be angiogenic in HOME cells.

All-trans-retinoic Acid-dependent Inhibition of E-Cadherin-based Cell Adhesion with Concomitant Dephosphorylation of β-Catenin in Metastatic Human Renal Carcinoma Cells

We previously described an in vitro invasion assay model, using a monolayer of vascular endothelial cells grown on collagen gel, that mimics the metastatic abilities of the highly metastatic human renal carcinoma cell lines, MM‐1,3 and 8 and their poorly metastatic counterparts, SN12C and Q‐8. MM‐1, 3 and 8 cells were observed to penetrate the monolayer of vascular endothelial cells and grew in a spreading or scattering manner with loose cell‐cell contact on collagen gel or on vascular endothelial cells. SN12C and Cl‐8 cells failed to penetrate and grew in a clustering manner with tight cell‐cell contact. Treatment with all‐trans‐retinoic acid (ATRA) at non‐toxic concentrations induced clustering or growth of MM‐1, 3 and 8 cells on collagen gel or on vascular endothelial cells with tight cell‐cell contact, and inhibited penetration. The clustering induced by ATRA was virtually blocked in the presence of anti‐E cadherin antibody. E‐Cadherin and β‐catenin were each localized mainly at the cell‐cell adherent junctions of colonizing cell populations that had been treated with ATRA. While the cellular levels of E‐cadherin and β‐catenin did not change significantly following ATRA treatment, the tyrosine residue of β‐catenin was rapidly dephosphorylated. The concomitant administration of Na vanadate, an inhibitor of tyrosine dephosphorylase, inhibited both the ATRA‐induced clustering and the dephosphorylation of β‐catenin tyrosine. ATRA‐induced clustering of MM‐3 cells may be linked to the state of tyrosine phosphorylation of β‐catenin.

A novel germline mutation of MSH2 in a hereditary nonpolyposis colorectal cancer patient with liposarcoma.

BACKGROUND:One of the clinical features of hereditary nonpolyposis colorectal cancer (HNPCC) is a high incidence of multiple primary neoplasms arising in various organs including the gastrointestinal and genitourinary tracts. Among extracolonic tumors, a limited number of soft tissue sarcomas associated with HNPCC have been reported, and the mechanism underlying liposarcoma in HNPCC patients remains unclear.AIM:We herein report the case of a HNPCC patient with liposarcoma, with the goal of elucidating the involvement of a mismatch repair deficiency in the tumor.METHODS AND RESULTS:A 40-yr-old Japanese patient, who had a past history of adenocarcinoma of the rectum and transitional cell carcinoma of the urinary bladder, developed a liposarcoma in his left thigh. Although his family history did not fulfill the revised Amsterdam criteria, his blood sample was subjected to genetic testing. Direct sequencing of the genomic DNA from the blood identified an AT deletion at codon 677 in exon 13 of hMSH2, a pathogenic mutation that has not been reported before. The expression of MSH2 in the liposarcoma and rectal cancer of the patient was analyzed by immunohistochemistry, which revealed loss of MSH2 expression in the tumors. To investigate whether the loss of MSH2 was a common feature of liposarcoma, we examined the MSH2 expression in an additional two sporadic liposarcomas, both of which were stained with anti-MSH2 antibody.CONCLUSION:We identified a novel pathogenic germline mutation of MSH2 in an HNPCC patient. Since an immunohistochemical analysis showed no nuclear staining for MSH2 protein in the liposarcoma as well as the rectal cancer, the loss of wild-type MSH2 protein was thus considered to possibly play a role in the development of liposarcoma in HNPCC patients.