Risk of idiopathic normal pressure hydrocephalus in older inpatients with schizophrenia.

BACKGROUND: The mean age of inpatients with schizophrenia has gradually increased in Japan and the risk of fracture in older schizophrenia patients is elevated. One possible cause may be idiopathic normal pressure hydrocephalus (iNPH). The present study aimed to evaluate the prevalence and symptoms of iNPH in older inpatients with schizophrenia. METHODS: We prospectively examined older inpatients with schizophrenia (N = 21, mean age = 70.5 ± 5.9) in a psychiatric ward. We evaluated iNPH symptoms using the idiopathic Normal-Pressure Hydrocephalus Grading Scale (iNPHGS), Timed Up-and-Go test (TUG), Gait Status Scale (GSS), Mini-Mental State Examination (MMSE), and Neuropsychiatric Inventory (NPI). We also evaluated symptoms of schizophrenia using the Brief Psychiatric Rating Scale (BPRS) and Drug-Induced Extrapyramidal Symptoms Scale (DIEPSS). We conducted cerebrospinal fluid (CSF) tap tests for patients with possible-iNPH. RESULTS: In total, three (14.3%) patients were diagnosed with possible iNPH: age, GS-Gait, GS-Cognition, TUG, 10-meter walking test, GSS, and DIEPSS were significantly increased in these compared to patients without iNPH; however, GS-Urine, MMSE, NPI, and BPRS did not differ significantly. Probable iNPH was diagnosed for two (9.5%) patients because of positive CSF tap tests. CONCLUSION: The prevalence of possible and probable iNPH in older patients with schizophrenia was much higher than that reported for older people without mental illness. Of the symptoms evaluated with the tests employed, only gait disturbances, particularly walking speed, distinguished schizophrenia patients with iNPH. These findings suggest that we should pay more attention to the possibility of iNPH in older patients with schizophrenia.

The effect of granulocyte and monocyte adsorptive apheresis on serum cytokine levels in patients with ulcerative colitis.

BACKGROUND: Granulocyte and monocyte adsorptive apheresis (GMA) with an Adacolumn has been reported to be effective as induction therapy in ulcerative colitis (UC). However, the effects of GMA on serial changes in cytokine levels have not been well characterized. We therefore, investigated cytokine levels in UC patients before and after treatment with GMA. A total of 16 patients with active UC, 10 men, and six women, mean age, 42.6 years were included. Fourteen patients had total colitis and two patients had left-sided colitis. The study included nine patients with a chronic intermittent course, six with a chronic continuous course and one with a single episode. The duration of each GMA session was 60 min at a flow rate of 30 mL/min as per study protocol. Serum levels of 17 cytokines were determined simultaneously using a Bio-Plex suspension array system before and after treatment with GMA. Serum interleukin (IL)-10 and macrophage inflammatory protein-1ß levels were increased significantly in UC patients after GMA treatment compared to pre-treatment levels (P < 0.05). In particular, GMA treatment caused a significant increase in serum IL-10 levels compared to pre-treatment in patients with total colitis or with a chronic intermittent UC course. In conclusion, this investigation showed that GMA was associated with a marked increase in serum level of the anti-inflammatory cytokine, IL-10. The rise in circulating IL-10 is interesting, and potentially a significant factor in the efficacy of GMA in patients with inflammatory bowel diseases.

Reactive astrocytes promote the metastatic growth of breast cancer stem-like cells by activating Notch signalling in brain.

Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1ß which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.

miR-7 suppresses brain metastasis of breast cancer stem-like cells by modulating KLF4.

Despite significant improvement in survival rates of patients with breast cancer, prognosis of metastatic disease is still dismal. Cancer stem-like cells (CSC) are considered to play a role in metastatic progression of breast cancer; however, the exact pathologic role of CSCs is yet to be elucidated. In this report, we found that CSCs (CD24(-)/CD44(+)/ESA(+)) isolated from metastatic breast cell lines are significantly more metastatic than non-CSC populations in an organ-specific manner. The results of our microRNA (miRNA) profile analysis for these cells revealed that CSCs that are highly metastatic to bone and brain expressed significantly lower level of miR-7 and that this miRNA was capable of modulating one of the essential genes for induced pluripotent stem cell, KLF4. Interestingly, high expression of KLF4 was significantly and inversely correlated to brain but not bone metastasis-free survival of patients with breast cancer, and we indeed found that the expression of miR-7 significantly suppressed the ability of CSCs to metastasize to brain but not to bone in our animal model. We also examined the expression of miR-7 and KLF4 in brain-metastatic lesions and found that these genes were significantly down- or upregulated, respectively, in the tumor cells in brain. Furthermore, the results of our in vitro experiments indicate that miR-7 attenuates the abilities of invasion and self-renewal of CSCs by modulating KLF4 expression. These results suggest that miR-7 and KLF4 may serve as biomarkers or therapeutic targets for brain metastasis of breast cancer.

Colonic mucosa-associated lymphoid tissue lymphoma.

Colonic mucosa-associated lymphoid tissue (MALT) lymphomas are rare and a definitive treatment has not been established. Solitary or multiple, elevated or polypoid lesions are the usual appearances of MALT lymphoma in the colon and sometimes the surface may reveal abnormal vascularity. In this paper we report our experience with four cases of colonic MALT lymphoma and review the relevant literature. The first patient had a smooth elevated lesion in the rectum and histopathologic examination of the biopsy from the lesion showed centrocyte-like cells infiltrating the lamina propria. Endoscopic ultrasonography (EUS) revealed thickening of the submucosa and muscularis propria. The patient underwent radiation therapy, and 9 months later a repeat colonoscopy showed complete resolution of the lesion. In case 2, colonoscopy showed a polyp in the cecum; the biopsy was diagnostic of MALT lymphoma. EUS detected a hypoechoic lesion confined to the mucosal layer of the colonic wall. The patient underwent endoscopic mucosal resection of the lesion and after 6 years of follow-up there was no evidence of recurrence. The third patient had a sessile elevated lesion in the sigmoid colon for which she underwent sigmoidectomy. Pathological examination of the surgical specimen was suggestive of MALT lymphoma. The last patient had a smooth elevated lesion in the rectum and magnification endoscopy showed irregular vascular pattern. The patient underwent endoscopic submucosal dissection, and biopsy examination showed the tumor to be MALT lymphoma. Although rare, awareness of MALT lymphoma of the colon is important to evaluate the patient appropriately and to plan further management.

Unusual Manifestation of Gastric Helicobacter pylori Infection.

Infection with Helicobacter pylori (HP) is common in many parts of the world. While most patients are asymptomatic, it causes peptic ulcer disease and malignancy in some of them. Other rare conditions have occasionally been reported in association with this infection. We report a case of hypertrophic gastropathy caused by HP in a 52-year-old asymptomatic patient. He was found to have marked enlargement of the gastric mucosal folds on radiological imaging and endoscopy. A gastric mucosal biopsy showed HP colonization associated with neutrophilic inflammation. After exclusion of neoplasia, other infections and infiltrative disorders, HP was thought to be the cause of the gastric fold hypertrophy. The patient responded well to HP eradication therapy, with normalization of the gastric mucosal folds. HP infection should be considered in the differential diagnosis of hypertrophic gastropathy and treated accordingly.

N-myc downstream regulated gene 1 modulates Wnt-ß-catenin signalling and pleiotropically suppresses metastasis.

Wnt signalling has pivotal roles in tumour progression and metastasis; however, the exact molecular mechanism of Wnt signalling in the metastatic process is as yet poorly defined. Here we demonstrate that the tumour metastasis suppressor gene, NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signalling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumour cells. Importantly, restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumour cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a novel negative master regulator of Wnt signalling during the metastatic progression, which opens an opportunity to define a potential therapeutic target for metastatic disease.

Hyaluronan synthase HAS2 promotes tumor progression in bone by stimulating the interaction of breast cancer stem-like cells with macrophages and stromal cells.

The molecular mechanisms that operate within the organ microenvironment to support metastatic progression remain unclear. Here, we report that upregulation of hyaluronan synthase 2 (HAS2) occurs in highly metastatic breast cancer stem-like cells (CSC) defined by CD44(+)/CD24(-)/ESA(+) phenotype, where it plays a critical role in the generation of a prometastatic microenvironment in breast cancer. HAS2 was critical for the interaction of CSCs with tumor-associated macrophages (TAM), leading to enhanced secretion of platelet-derived growth factor-BB from TAMs, which then activated stromal cells and enhanced CSC self-renewal. Loss of HAS2 in CSCs or treatment with 4-methylumbelliferone, an inhibitor of HAS, which blocks hyaluronan production, drastically reduced the incidence and growth of metastatic lesions in vitro or in vivo, respectively. Taken together, our findings show a critical role of HAS2 in the development of a prometastatic microenvironment and suggest that HAS2 inhibitors can act as antimetastatic agents that disrupt a paracrine growth factor loop within this microenvironment.

Bone morphogenetic protein 7 in dormancy and metastasis of prostate cancer stem-like cells in bone.

Metastatic disease is the major cause of cancer deaths, and recurrent tumors at distant organs are a critical issue. However, how metastatic tumor cells become dormant and how and why tumors recur in target organs are not well understood. In this study, we demonstrate that BMP7 (bone morphogenetic protein 7) secreted from bone stromal cells induces senescence in prostate cancer stem-like cells (CSCs) by activating p38 mitogen-activated protein kinase and increasing expression of the cell cycle inhibitor, p21, and the metastasis suppressor gene, NDRG1 (N-myc downstream-regulated gene 1). This effect of BMP7 depended on BMPR2 (BMP receptor 2), and BMPR2 expression inversely correlated with recurrence and bone metastasis in prostate cancer patients. Importantly, this BMP7-induced senescence in CSCs was reversible upon withdrawal of BMP7. Furthermore, treatment of mice with BMP7 significantly suppressed the growth of CSCs in bone, whereas the withdrawal of BMP7 restarted growth of these cells. These results suggest that the BMP7-BMPR2-p38-NDRG1 axis plays a critical role in dormancy and recurrence of prostate CSCs in bone and suggest a potential therapeutic utility of BMP7 for recurrent metastatic disease.

KAI1 gene is engaged in NDRG1 gene-mediated metastasis suppression through the ATF3-NFkappaB complex in human prostate cancer.

NDRG1 and KAI1 belong to metastasis suppressor genes, which impede the dissemination of tumor cells from primary tumors to distant organs. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of the KAI1 gene. In this report, we found that ectopic expression of NDRG1 was able to augment endogenous KAI1 gene expression in prostate cancer cell lines, whereas silencing NDRG1 was accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of the KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFκB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional downstream target of the NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through an intrinsic transcriptional cascade.

Resveratrol suppresses growth of cancer stem-like cells by inhibiting fatty acid synthase.

Resveratrol is a natural polyphenolic compound and has been shown to exhibit cardio-protective as well as anti-neoplastic effects on various types of cancers. However, the exact mechanism of its anti-tumor effect is not clearly defined. Resveratrol has been shown to have strong hypolipidemic effect on normal adipocytes and as hyper-lipogenesis is a hallmark of cancer cell physiology, the effect of resveratrol on lipid synthesis in cancer stem-like cells (CD24(-)/CD44(+)/ESA(+)) that were isolated from both ER+ and ER- breast cancer cell lines was examined. The authors found that resveratrol significantly reduced the cell viability and mammosphere formation followed by inducing apoptosis in cancer stem-like cells. This inhibitory effect of resveratrol is accompanied by a significant reduction in lipid synthesis which is caused by the down-regulation of the fatty acid synthase (FAS) gene followed by up-regulation of pro-apoptotic genes, DAPK2 and BNIP3. The activation of apoptotic pathway in the cancer stem-like cells was suppressed by TOFA and by Fumonisin B1, suggesting that resveratrol-induced apoptosis is indeed through the modulation of FAS-mediated cell survival signaling. Importantly, resveratrol was able to significantly suppress the growth of cancer stem-like cells in an animal model of xenograft without showing apparental toxicity. Taken together, the results of this study indicate that resveratrol is capable of inducing apoptosis in the cancer stem-like cells through suppression of lipogenesis by modulating FAS expression, which highlights a novel mechanism of anti-tumor effect of resveratrol.

RhoC promotes metastasis via activation of the Pyk2 pathway in prostate cancer.

RhoC is a member of the Ras-homologous family of genes which have been implicated in tumorigenesis and tumor progression. However, the exact role of RhoC is controversial and is yet to be clarified. We have examined the effect of RhoC on prostate tumor cells and found that RhoC had no effect on cell proliferation in vitro or on tumor growth in mice. However, RhoC significantly enhanced the metastatic ability of the tumor cells in these animals, suggesting that RhoC affects only the metastasis but not the growth of prostate tumor cells. The results of our immunohistochemical analyses on tumor specimens from 63 patients with prostate cancer indicate that RhoC expression had no significant correlation with Gleason grade. However, the expression of RhoC showed significant positive correlation with both lymph node and distant metastasis, and it was inversely correlated with patient survival. We also found that RhoC significantly augmented the invasion and motility of prostate tumor cells by activating matrix metalloproteinases 2 and 9 (MMP2 and MMP9) in vitro. The results of our antibody array analysis for signal molecules revealed that RhoC significantly activated kinases including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt, and Pyk2. Inhibition of Pyk2 kinase blocked the RhoC-dependent activation of FAK, MAPK, and Akt, followed by the suppression of MMP2 and MMP9. Inhibitors of both MAPK and Akt also significantly blocked the activities of these MMPs. Therefore, our results indicate that RhoC promotes tumor metastasis in prostate cancer by sequential activation of Pyk2, FAK, MAPK, and Akt followed by the up-regulation of MMP2 and MMP9, which results in the stimulation of invasiveness of tumor cells.

Fatty acid synthase gene is up-regulated by hypoxia via activation of Akt and sterol regulatory element binding protein-1.

The fatty acid synthase (FAS) gene is significantly up-regulated in various types of cancers, and blocking the FAS expression results in apoptosis of tumor cells. Therefore, FAS is considered to be an attractive target for anticancer therapy. However, the molecular mechanism by which the FAS gene is up-regulated in tumor cells is poorly understood. We found that FAS was significantly up-regulated by hypoxia, which was also accompanied by reactive oxygen species (ROS) generation in human breast cancer cell lines. The FAS expression was also activated by H(2)O(2), whereas N-acetyl-L-cystein, a ROS inhibitor, suppressed the expression. We also found that the hypoxia significantly up-regulated sterol regulatory-element binding protein (SREBP)-1, the major transcriptional regulator of the FAS gene, via phosphorylation of Akt followed by activation of hypoxia-inducible factor 1 (HIF1). Moreover, our results of reporter assay and chromatin immunoprecipitation analysis indicate that SREBP-1 strongly bound to the SREBP binding site/E-box sequence on the FAS promoter under hypoxia. In our xenograft mouse model, FAS was strongly expressed in the hypoxic regions of the tumor. In addition, our results of immunohistochemical analysis for human breast tumor specimens indicate that the expressions of both FAS and SREBP-1 were colocalized with hypoxic regions in the tumors. Furthermore, we found that hypoxia-induced chemoresistance to cyclophosphamide was partially blocked by a combination of FAS inhibitor and cyclophosphamide. Taken together, our results indicate that FAS gene is up-regulated by hypoxia via activation of the Akt and HIF1 followed by the induction of the SREBP-1 gene, and that hypoxia-induced chemoresistance is partly due to the up-regulation of FAS.

The tumor metastasis suppressor gene Drg-1 down-regulates the expression of activating transcription factor 3 in prostate cancer.

The tumor metastasis suppressor gene Drg-1 has been shown to suppress metastasis without affecting tumorigenicity in immunodeficient mouse models of prostate and colon cancer. Expression of Drg-1 has also been found to have a significant inverse correlation with metastasis or invasiveness in various types of human cancer. However, how Drg-1 exerts its metastasis suppressor function remains unknown. In the present study, to elucidate the mechanism of action of the Drg-1 gene, we did a microarray analysis and found that induction of Drg-1 significantly inhibited the expression of activating transcription factor (ATF) 3, a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. We also showed that Drg-1 attenuated the endogenous level of ATF3 mRNA and protein in prostate cancer cells, whereas Drg-1 small interfering RNA up-regulated the ATF3 expression. Furthermore, Drg-1 suppressed the promoter activity of the ATF3 gene, indicating that Drg-1 regulates ATF3 expression at the transcriptional level. Our immunohistochemical analysis on prostate cancer specimens revealed that nuclear expression of ATF3 was inversely correlated to Drg-1 expression and positively correlated to metastases. Consistently, we have found that ATF3 overexpression promoted invasiveness of prostate tumor cells in vitro, whereas Drg-1 suppressed the invasive ability of these cells. More importantly, overexpression of ATF3 in prostate cancer cells significantly enhanced spontaneous lung metastasis of these cells without affecting primary tumorigenicity in a severe combined immunodeficient mouse model. Taken together, our results strongly suggest that Drg-1 suppresses metastasis of prostate tumor cells, at least in part, by inhibiting the invasive ability of the cells via down-regulation of the expression of the ATF3 gene.

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.

Mechanism of apoptosis induced by the inhibition of fatty acid synthase in breast cancer cells.

Fatty acid synthase (FAS) has been found to be overexpressed in a wide range of epithelial tumors, including breast cancer. Pharmacologic inhibitors of FAS cause apoptosis of breast cancer cells and result in decreased tumor size in vivo. However, how the inhibition of FAS induces apoptosis in tumor cells remains largely unknown. To understand the apoptotic pathway resulting from direct inhibition of FAS, we treated breast tumor cells with or without FAS small interfering RNA (siRNA) followed by a microarray analysis. Our results indicated that the proapoptotic genes BNIP3, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and death-associated protein kinase 2 (DAPK2) were significantly up-regulated on direct inhibition of the FAS gene. We also found that the knockdown of FAS expression significantly increased ceramide level in the tumor cells, and this increase was abrogated by acetyl-CoA carboxylase inhibitor. In addition, carnitine palmitoyltransferase-1 (CPT-1) inhibitor up-regulated the ceramide and BNIP3 levels in these cells, whereas treatment of tumor cells with FAS siRNA in the presence of a ceramide synthase inhibitor abrogated the up-regulation of BNIP3 and inhibited apoptosis. Furthermore, we found that treatment of cells with BNIP3 siRNA significantly counteracted the effect of FAS siRNA-mediated apoptosis. Consistent with these results, a significant inverse correlation was observed in the expression of FAS and BNIP3 in clinical samples of human breast cancer. Collectively, our results indicate that inhibition of FAS in breast cancer cells causes accumulation of malonyl-CoA, which leads to inhibition of CPT-1 and up-regulation of ceramide and induction of the proapoptotic genes BNIP3, TRAIL, and DAPK2, resulting in apoptosis.

FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis.

Fatty acid synthase (FAS), a key enzyme of the fatty acid biosynthetic pathway, has been shown to be overexpressed in various types of human cancer and is, therefore, considered to be an attractive target for anticancer therapy. However, the exact mechanism of overexpression of the FAS gene in tumor cells is not well understood. In this report, we demonstrate that the expression of the tumor suppressor gene PTEN has a significant inverse correlation with FAS expression in the case of prostate cancer in the clinical setting, and inhibition of the PTEN gene leads to the overexpression of FAS in vitro. We also found that the combination of the expression status of these two genes is a better prognostic marker than either gene alone. Furthermore, our results indicate that the specific inhibition of FAS gene by siRNA leads to apoptosis of prostate tumor cells, and inhibition of PI 3-kinase pathway synergizes with FAS siRNA to enhance tumor cell death. These results provide a strong rationale for exploring the therapeutic use of an inhibitor of the PTEN signaling pathway in conjunction with the FAS siRNA to inhibit prostate tumor growth.

PTEN up-regulates the tumor metastasis suppressor gene Drg-1 in prostate and breast cancer.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) has been shown to be inactivated in a wide variety of cancers, and the role of this gene as a tumor suppressor has been well established. On the other hand, results of recent animal studies as well as clinical evidence indicate that PTEN is also involved in tumor metastasis suppression. Although PTEN is known to play a key role in controlling cell growth and apoptosis, how PTEN exerts the metastasis suppressor function remains largely unknown. Recently, a microarray analysis identified the Drg-1 gene (differentiation related gene 1) as one of the potential targets of PTEN. The Drg-1 gene has been shown to suppress tumor metastasis in animal models of prostate and colon cancer, and the expression of this gene is significantly reduced with advancement of prostate and breast cancers in clinical setting. In this study, we explored the possibility that PTEN controls tumor metastasis by regulating the expression of the Drg-1 gene. Our results indicate that overexpression of PTEN significantly augments the endogenous expression of Drg-1 protein, whereas inhibition of PTEN by small interfering RNA decreases Drg-1 in a dose- and time-dependent manner. We also found that the control of the Drg-1 gene by PTEN seems to be at the transcriptional level, and that a phospho-Akt inhibitor restores the Drg-1 expression, indicating that PTEN controls Drg-1 by an Akt-dependent pathway. Consistent with these results, our immunohistochemical analysis revealed that PTEN expression correlates significantly with Drg-1 in both prostate and breast cancer cases. Furthermore, combination of the two markers, PTEN and Drg-1, emerged as a significantly better predictor of prostate and breast cancer patient survival than either marker alone.

Role of the putative tumor metastasis suppressor gene Drg-1 in breast cancer progression.

The differentiation-related gene-1 (Drg-1) was first identified as a gene strongly upregulated by induction of differentiation in colon carcinoma cells in vitro, and later the same gene was shown to suppress tumorigenicity of human bladder cancer cells in vivo. On the other hand, we and others have demonstrated that the Drg-1 gene suppresses prostate and colon cancer metastases in mouse models. In the context of such potential organ-specific differential function of the Drg-1 gene, the present study was designed to clarify the expression status, regulation and function of Drg-1 in the case of human breast cancer. We found that the expression of the Drg-1 protein was significantly reduced in breast tumor cells, particularly in patients with lymph node or bone metastasis as compared to those with localized breast cancer. Drg-1 expression also exhibited significant inverse correlation with the disease-free survival rate of patients and emerged as an independent prognostic factor. The downregulation of the Drg-1 gene appeared to be largely at the RNA level, and the DNA methylation inhibitor, 5-Azacytidine, significantly elevated the Drg-1 gene expression in various breast tumor cell lines. Furthermore, we found that overexpression of the Drg-1 gene suppresses the invasiveness of breast cancer cells in vitro, and this suppression was also achieved by treatment of cells with 5-Azacytidine. Together, our results strongly suggest functional involvement of the Drg-1 gene in suppressing the metastatic advancement of human breast cancer.

The Drg-1 gene suppresses tumor metastasis in prostate cancer.

Drg-1 was previously identified (N. van Belzen et al., Lab. Investig., 77: 85-92, 1997) as a gene that was up-regulated by the induction of differentiation in a colon carcinoma cell line in vitro. Subsequently, this gene was found to be regulated by several factors including hypoxia, androgen, p53, and N-myc. Recently, Drg-1 has also been shown to be involved in tumor progression in animals, although the clinical significance of its involvement remains to be investigated. To clarify the functional role of Drg-1 in prostate cancer, we examined a clinical archive of cancer specimens for the expression of Drg-1 by immunohistochemistry. We found that the expression of Drg-1 had a significant inverse correlation with the Gleason grading and the overall survival rate of patients. In particular, the gene expression in patients with lymph node or bone metastasis was significantly reduced as compared with those with localized prostate cancer, suggesting that the function of Drg-1 is negatively involved in metastatic progression of the disease. To further clarify the function of this gene in the advancement of prostate cancer, a spontaneous metastasis assay was performed in a severe combined immunodeficient (SCID) mouse model. We found that Drg-1 almost completely inhibited lung colonization of highly metastatic prostate cancer cells without affecting the growth of the primary tumors. These results strongly suggest that Drg-1 is a candidate metastasis suppressor gene for prostate cancer and may serve as a useful prognostic marker.