Downregulation of microRNA‐15b by hepatitis B virus X enhances hepatocellular carcinoma proliferation via fucosyltransferase 2‐induced Globo H expression

Globo H, a cancer‐associated carbohydrate antigen, is highly expressed in various types of cancers. However, the role of Globo H in hepatocellular carcinoma (HCC) remains elusive. In our study, we performed glycan microarray analysis of 134 human serum samples to explore anti‐Globo H antibody changes and found that Globo H is upregulated in hepatitis B virus (HBV)‐positive HCC. Similarly, immunohistochemistry showed that Globo H expression was higher in tumors compared to normal tissues. In addition, fucosyltransferase 2 (FUT2), the main synthetic enzyme of Globo H, was also increased in HCC cells overexpressing HBV X protein (HBX). HBX plays an important role in promoting cell proliferation and may be related to increased levels of FUT2 and Globo H. Furthermore, using microRNA profiling, we observed that microRNA‐15b (miR‐15b) was downregulated in patients with HCC and confirmed association of FUT2 expression with expression of its product, Globo H. Therefore, our results suggest that HBX suppressed the expression of miR‐15b, which directly targeted FUT2 and then increased levels of Globo H to enhance HCC cell proliferation. Additionally, proliferation of HBX‐overexpressing HCC cells was significantly inhibited by treatment with Globo H antibody in vitro. In xenograft animal experiments, we found that overexpression of miR‐15b effectively suppressed tumor growth. The newly identified HBX/miR‐15b/FUT2/Globo H axis suggests one possible molecular mechanism of HCC cell proliferation and represents a new potential therapeutic target for HCC treatment.     

Susceptibility of rat colon carcinoma cells to lymphokine activated killer-mediated cytotoxicity is decreased by alpha1,2-fucosylation

The presence of alpha1,2-fucosylated glycans at the surface of rat colon carcinoma cells has been associated with an increased tumorigenicity and resistance to natural killer/lymphokine activated killer (NK/LAK) cytotoxicity. We now report that transfection of rat alpha1,2-fucosyltransferases cDNA (FTA and FTB) into REG cells, which are spontaneously devoid of this enzymatic activity, allows expression of histo-blood group H antigen and increases their resistance to LAK, but not NK cell lysis. Conversely, transfection of PRO cells, which spontaneously express alpha1, 2-fucosyltransferase activity, with the FTA cDNA in the antisense orientation decreases expression of the H antigen together with their resistance to LAK cell lysis, but again, not to NK cell lysis. Furthermore, REG cells that are rejected by immunocompetent syngeneic rats are similarly rejected by rats depleted of NK cells by antibody 3.2.3, directed against the NKR-P1 molecule. Thus, the rejection of REG cells by immunocompetent rats and their earlier reported increased tumorigenicity after transfection with an alpha1, 2-fucosyltransferase cDNA cannot be ascribed to NK cell sensitivity or resistance, respectively. The increased resistance to LAK cell lysis, however, may be relevant to tumor progression.     

α1，2-岩藻糖转移酶基因转染对卵巢癌细胞中HER2/neu表达及活性的影响

目的:探讨α1,2-岩藻糖转移酶（α1,2-fucosyltransferase,FUT1）基因转染对人卵巢癌细胞中HER2/neu表达及活性的影响。方法:分别应用Real time PCR、免疫细胞化学染色和Western blot方法检测转染前后卵巢癌细胞中HER2/neu在基因、蛋白水平上的表达和磷酸化情况,利用免疫共沉淀方法检测HER2/neu蛋白上是否有Lewis(y) 结构。结果:Real time PCR结果显示转染后细胞中HER2/neu mRNA表达明显增高。免疫细胞化学染色、免疫共沉淀结合Western blot检测到转染后卵巢癌细胞中HER2/neu蛋白和Lewis(y)抗原的表达均较转染前显著增加,HER2/neu蛋白上有Lewis(y)抗原结构。Western blot结果显示基因转染后HER2/neu的酪氨酸磷酸化水平显著升高。结论:Lewis(y)抗原是HER2/neu结构上的一部分,其表达增加不但促进卵巢癌细胞中HER2/neu的表达,还激活了HER2/neu受体酪氨酸激酶。     

Changes in the invasive and metastatic capacities of HT-29/M3 cells induced by the expression of fucosyltransferase 1

Lewis antigens are terminal fucosylated oligosaccharides synthesized by the sequential action of several glycosyltransferases. The fucosyltransferases are the enzymes responsible for the addition of terminal fucose to precursor oligosaccharides attached to proteins or lipids. These oligosaccharides, defined as cell surface markers, have been implicated in different types of intercellular interactions and in adhesion and invasion processes. Transfection of HT-29/M3 colon cancer cells with the full length of human fucosyltransferase (FUT1), induces the synthesis of H type 2 and Lewis y antigens, associated with a decrease of sialyl-Lewis x. The capacity to develop primary tumors when cells were injected intrasplenically was similar in parental and FUT1-transfected cells, but the capacity to colonize the liver after spleen removal was significantly reduced in M3/FUT1 transfected cells. These results indicate that the expression of FUT1 induces changes in the metastatic capacity of HT-29/M3 colon cancer cells, as a consequence of the altered expression pattern of type 2 Lewis antigens. Also, an association between MUC5AC expression and the degree of gland differentiation in both primary splenic tumors and hepatic metastases was detected.     

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

About 90% of human pancreatic cancers carry K‐ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti‐sense oligonucleotides targeting K‐ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K‐ras codon 12 point mutations by PCR‐RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K‐ras point mutation, i.e.,HuP‐T1, HuP‐T3 and PANC‐1, and 1 without mutation, BxPC‐3, were used for the experiments. Seventeen mer anti‐sense oligonucleotides were designed, targeting the point mutation of K‐ras codon 12, and transfected into the cells by the liposome‐mediated method. Cell‐growth activities were estimated by MTT assay. Levels of K‐ras mRNA expression were determined using quantitative RT‐PCR, and K‐ras p21 protein synthesis was evaluated with Western blotting. Mutation‐matched anti‐sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC‐3, by suppressing K‐ras mRNA expression and K‐ras p21 protein synthesis. Moreover, mutation‐matched anti‐sense oligonucleotides showed stronger anti‐proliferative effects than did mutation‐mismatched ones. Our results suggest that anti‐sense therapy specific to point mutations of K‐ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells. Int. J. Cancer 80:553–558, 1999. © 1999 Wiley‐Liss, Inc.     

Role of target antigen in bispecific‐antibody‐mediated killing of human glioblastoma cells: A pre‐clinical study

Bispecific antibodies (bsAbs) directed to tumor‐associated antigens and to receptors mediating T‐cell activation, such as the TCR/CD3 complex and the co‐stimulatory CD28 molecule, are capable of activating T cells at the surface of tumor cells, resulting in tumor‐cell killing. Here we report the pre‐clinical characterization of bispecific‐antibody fragments (bsFab₂) directed to 2 different glioblastoma‐associated antigens: the EGF receptor (EGFR) and a chondroitin‐sulfate proteoglycan (CSPG). Using cultured glioblastoma cells expressing both target antigens, we found that the ability of anti‐tumor × anti‐CD28 bsFab₂ to mediate “targeted T‐cell co‐stimulation” is superior for constructs targeting the CSPG molecule, correlating with an approximately 6‐fold higher expression level of this antigen on the cell surface. In contrast, bsFab₂ triggering CD3 are more effective if they contain EGFR‐target specificity. This indicates that the activity of anti‐tumor × anti‐CD3 constructs critically depends on properties of the antigen other than its expression level on the cell surface, e.g., its mobility in the membrane. These findings prompted us to use EGFR‐targeting bsFab₂ in an ongoing clinical trial with glioma patients. Int. J. Cancer 80:612–616, 1999. © 1999 Wiley‐Liss, Inc.     

Exogenous introduction of tissue inhibitor of metalloproteinase 2 reduces accelerated growth of TGF‐β‐disrupted diffuse‐type gastric carcinoma

Diffuse‐type gastric carcinoma is characterized by rapid progression and poor prognosis. High expression of transforming growth factor (TGF)‐β and thick stromal fibrosis are observed in this type of gastric carcinoma. We have previously shown that disruption of TGF‐β signaling via introduction of a dominant negative form of the TGF‐β type II receptor (dnTβRII) into diffuse‐type gastric cancer cell lines, including OCUM‐2MLN, caused accelerated tumor growth through induction of tumor angiogenesis in vivo. In the present study, we show that TGF‐β induces upregulation of expression of tissue inhibitor of metalloproteinase 2 (TIMP2) in the OCUM‐2MLN cell line in vitro, and that expression of TIMP2 is repressed by dnTβRII expression in vivo. Transplantation of the OCUM‐2MLN cells to nude mice exhibited accelerated tumor growth in response to dnTβRII expression, which was completely abolished when TIMP2 was coexpressed with dnTβRII. Although the blood vessel density of TIMP2‐expressing tumors was only slightly decreased, the degree of hypoxia in tumor tissues was significantly increased and pericytes covering tumor vasculature were decreased by TIMP2 expression in OCUM‐2MLN cells, suggesting that the function of tumor vasculatures was repressed by TIMP2 and consequently tumor growth was reduced. These findings provide evidence that one of the mechanisms of the increase in angiogenesis in diffuse‐type gastric carcinoma is the downregulation of the anti‐angiogenic protein TIMP2. (Cancer Sci 2010; 101: 2398–2403)     

Synergistic anti‐tumor efficacy of immunogenic adenovirus ONCOS‐102 (Ad5/3‐D24‐GM‐CSF) and standard of care chemotherapy in preclinical mesothelioma model

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1‐year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed‐Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression‐free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS‐102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM‐CSF. The safety and immune activating properties of ONCOS‐102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS‐102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS‐102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti‐tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti‐tumor activity in the mesothelioma mouse model, ONCOS‐102 was able to slow down tumor growth. Interestingly, a synergistic anti‐tumor effect was seen when ONCOS‐102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS‐102 in combination with first‐line chemotherapy in patients suffering from malignant mesothelioma.     

Identification of mitochondrial F1F0‐ATP synthase interacting with galectin‐3 in colon cancer cells

To evaluate the effect of galectin‐3 in cell cycle regulation of colon cancer cells, we looked for binding molecules interacting with galectin‐3 and examined the changes in cell cycle by suppressing galectin‐3 and the binding molecule. To identify target molecules interacting with galectin‐3, we analyzed immunoprecipitate of the anti‐galectin‐3 antibody obtained from human colon cancer cell line, using matrix‐assisted laser desorption ionization‐mass spectrometry. We validated subcellular localization of galectin‐3 and ATP synthase identified, and ATP synthase activity was determined in the presence of galectin‐3. Cell cycle regulation was monitored after galectin‐3 siRNA transfection. ATP synthase b‐subunit was identified in immunoprecipitate of the anti‐galectin‐3 antibody. Galectin‐3 and ATP synthase were co‐isolated in the inner membrane vesicles of mitochondria. Galectin‐3 has an inhibitory activity against ATP synthase, and intracellular ATP content showed increasing tendency after galectin‐3 suppression. Suppression of galectin‐3 resulted in G0/G1 progression of human colon cancer cells arrested at S, S/G2 and G2/M phase in the presence of doxorubicin, and etoposide or nocodazole, respectively. Compared to cells in which ATP synthase d‐subunit was suppressed alone, sub‐G1 fraction caused by etoposide or nocodazole was decreased in cells with galectin‐3 suppression alone. In conclusion, galectin‐3 co‐localized with ATP synthase in the inner membrane of mitochondria and has an inhibitory effect on ATP synthase in human colon cancer cells. In the presence of cell cycle synchronizing drugs, doxorubicin, etoposide, or nocodazole, suppression of galectin‐3 induced cell cycle progression to G0/G1 phase. (Cancer Sci 2008; 99: 1884–1891)     

Increased transforming growth factor‐α levels in human colon carcinoma cell lines over‐expressing protein kinase C

Transforming growth factor‐α (TGF‐α) is synthesized as a membrane‐bound precursor protein, pro‐TGF‐α, that is converted to a soluble form by 2 endoproteolytic cleavages. Several factors have been implicated in the regulation of the second rate‐limiting step, including protein kinase C (PKC). Earlier results indicated a potential role for the conventional class of PKC isozymes in the observed increase in TGF‐α in the conditioned media of 2 human colon carcinoma cell lines. The present study addresses the potential role of specific PKC isozymes in this process using sense and anti‐sense expression vectors for PKC isozymes. Two human colon carcinoma cell lines, HCT 116 and GEO, were transfected with plasmids, leading to the over‐expression of PKC‐α, ‐βI or ‐βII; and the secretion of TGF‐α into the conditioned medium was determined. Over‐expression of either PKC‐βI or PKC‐βII in these cell lines enhanced the levels of TGF‐α in the media 2‐ to 5‐fold. Over‐expression of PKC‐α did not alter the amount of TGF‐α in the media to a significant extent. Transfection of HCT 116 cells with the anti‐sense PKC‐βI cDNA resulted in a reduction in PKC‐βI protein expression. This was accompanied by a decrease in the amount of TGF‐α in the conditioned media. Our results indicate that modulation of PKC‐β protein levels alters the amount of TGF‐α found in the conditioned media from these colon carcinoma cells. Int. J. Cancer 80:72–77, 1999. © 1999 Wiley‐Liss, Inc.     

Role of α‐gal epitope/anti‐Gal antibody reaction in immunotherapy and its clinical application in pancreatic cancer

Pancreatic cancer is one of the most common causes of death from cancer. Despite the availability of various treatment modalities, such as surgery, chemotherapy and radiotherapy, the 5‐year survival remains poor. Although gemcitabine‐based chemotherapy is typically offered as the standard care, most patients do not survive longer than 6 months. Therefore, new therapeutic approaches are needed. The α‐gal epitope (Galα1‐3Galβ1‐4GlcNAc‐R) is abundantly synthesized from glycoproteins and glycolipids in non‐primate mammals and New World monkeys, but is absent in humans, apes and Old World monkeys. Instead, they produce anti‐Gal antibody (Ab) (forming approximately 1% of circulating immunoglobulins), which specifically interacts with α‐gal epitopes. Anti‐Gal Ab can be exploited in cancer immunotherapy as vaccines that target antigen‐presenting cells (APC) to increase their immunogenicity. Tumor cells or tumor cell membranes from pancreatic cancer are processed to express α‐gal epitopes. Subsequent vaccination with such processed cell membranes results in in vivo opsonization by anti‐Gal IgG in cancer patients. The interaction of the Fc portion of the vaccine‐bound anti‐Gal with Fcγ receptors of APC induces effective uptake of the vaccinating tumor cell membranes by the APC, followed by effective transport of the vaccinating tumor membranes to the regional lymph nodes, and processing and presentation of the tumor‐associated antigens. Activation of tumor‐specific B and T cells could elicit an immune response that in some patients is potent enough to eradicate the residual cancer cells that remain after completion of standard therapy. This review addresses these topics and new avenues of clinical importance related to this unique antigen/antibody system (α‐gal epitope/anti‐Gal Ab) and advances in immunotherapy in pancreatic cancer.     

Development of DS‐5573a: A novel afucosylated mAb directed at B7‐H3 with potent antitumor activity

B7‐H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti‐human B7‐H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu‐M30), and an afucosylated Hu‐M30 (DS‐5573a) was also generated. To assess the potency of DS‐5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7‐H3 proteins were expressed on various types of cancer cell lines broadly, and DS‐5573a binds to IgC1 and IgC2 domains of human B7‐H3. Antibody‐dependent cellular cytotoxicity activity of DS‐5573a was drastically enhanced against medium to high B7‐H3‐expressing cancer cell lines MDA‐MB‐231 and NCI‐H322. DS‐5573a also induced high antibody‐dependent cellular cytotoxicity activity against low B7‐H3‐expressing cancer cell line COLO205, whereas Hu‐M30 induced little activity against it. In addition, DS‐5573a was found to be a novel anti‐B7‐H3 antibody which showed antibody‐dependent cellular phagocytosis activity. Furthermore, DS‐5573a showed dose‐dependent and significant antitumor efficacy (0.03–3 mg/kg) in MDA‐MB‐231‐bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS‐5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7‐H3‐expressing tumors.     

Type V collagen and protein kinase C η down‐regulation in 8701‐BC breast cancer cells

We previously reported that ductal infiltrating carcinomas (d.i.c.) of the human breast display profound modifications of the stromal architecture, associated with anomalous collagen composition. Among the major alterations observed in the interstitial collagen, the relative increase of type V collagen content was detected. When type V collagen was used as an “in vitro” substrate for 8701‐BC d.i.c. cells, it appeared able to restrain cell growth, inhibit cell motility and invasion “in vitro”, and modify the expression levels of genes coding for apoptosis factors, caspases and stress response proteins. In the present paper we demonstrate that type V collagen induces the down‐regulation of protein kinase C η, an event that may be, at least in part, responsible of the previously‐reported modifications of cell morphology and growth rate, and that appears to be involved in the already‐observed changes of expression levels of genes encoding for anti‐ (Bcl‐2) and pro‐apoptotic factors (Bad, Dapk, Bcl‐Xs) and enzymes (caspase 5 and 8). © 2011 Wiley Periodicals, Inc.     

3‐Methylcholanthrene‐induced transforming growth factor‐β‐producing carcinomas,but not sarcomas,are refractory to regulatory T cell‐depletion therapy

Regulatory T cells (Tregs) are major immunosuppressors in tumor‐bearing hosts. Although Treg‐depletion therapy has been shown to induce a complete cure in tumor‐bearing mice, this treatment is not always successful. Using 3‐methylcholanthrene‐induced primary mouse tumors, we examined the distinct regulation of Treg‐mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)‐bearing mice compared with sarcoma‐bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)‐β, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg‐depletion therapy by treatment with anti‐CD25 mAb. The refractoriness of SCC against Treg‐depletion therapy was due to the rapid recovery of Tregs in SCC‐bearing mice compared with sarcoma‐bearing mice. However, combination treatment of anti‐TGF‐β mAb with anti‐CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC‐bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg‐depletion therapy using anti‐CD25 mAb treatment. We also proposed a novel Treg‐blocking combination therapy using anti‐CD25 mAb and anti‐TGF‐β mAb to induce a complete cure of tumor‐bearing hosts. (Cancer Sci 2010; 101: 855–861)