ARHI (DIRAS3), an imprinted tumour suppressor gene, binds to importins and blocks nuclear import of cargo proteins.

ARHI (aplasia Ras homologue member I; also known as DIRAS3) is an imprinted tumour suppressor gene, the expression of which is lost in the majority of breast and ovarian cancers. Unlike its homologues Ras and Rap, ARHI functions as a tumour suppressor. Our previous study showed that ARHI can interact with the transcriptional activator STAT3 (signal transducer and activator of transcription 3) and inhibit its nuclear translocation in human breast- and ovarian-cancer cells. To identify proteins that interact with ARHI in nuclear translocation, in the present study, we performed proteomic analysis and identified several importins that can associate with ARHI. To further explore this novel finding, we purified 10 GST (glutathione transferase)-importin fusion proteins (importins 7, 8, 13, beta1, alpha1, alpha3, alpha5, alpha6, alpha7 and mutant alpha1). Using a GST-pulldown assay, we found that ARHI can bind strongly to most importins; however, its binding is markedly reduced with an importin alpha1 mutant that contains an altered NLS (nuclear-localization signal) domain. In addition, an ARHI N-terminal deletion mutant exhibits greatly reduced binding to all importins compared with wild-type ARHI. In nuclear-import assays, the addition of ARHI blocked nuclear localization of phosphorylated STAT3. ARHI also inhibits the interaction of Ran-importin complexes with GFP (green fluorescent protein) fusion proteins that contain an NLS domain and a beta-like import receptor-binding domain, thereby blocking their nuclear localization. By conducting GST-pulldown assays, we found that ARHI could compete for Ran-importin binding. Thus ARHI-induced disruption of importin-binding to cargo proteins, including STAT3, could serve as an important regulatory mechanism that contributes to the tumour-suppressor function of ARHI.

The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

MUC16 expression during embryogenesis, in adult tissues, and ovarian cancer in the mouse.

Cancer antigen 125 (CA125) is an antigen that is elevated in the serum of women with ovarian carcinoma, but can also be detected in serum from healthy women. CA125 is expressed in 80% of human ovarian cancers, as well as in normal adult endometrium, lung, and amnion. The gene encoding human CA125 was identified as MUCIN16 (MUC16). A database search identified the orthologous mouse gene, Muc16. Reverse transcription-polymerase chain reaction and RNA in situ hybridization detected Muc16 transcripts in the surface epithelia of the upper respiratory tract, the mesothelia lining body cavities and the internal organs, as well as male and female reproductive organs, and the amnion. Antibodies raised against human MUC16 do not recognize mouse MUC16. Therefore, a rabbit anti-mouse polyclonal antibody against recombinant mouse MUC16 was generated. Immunohistochemistry using this anti-mouse MUC16 antibody revealed expression in the luminal epithelia of the trachea, the epithelia of the secretory glands in the oral cavity, the surface of the olfactory epithelia, as well as mesothelial cells lining body cavities (i.e., pleural, peritoneal, and pelvic cavities), and male and female reproductive organs. In addition, MUC16 protein was detected in other cell types, such as the surface epithelia of the cochlear duct and chief cells of the stomach, suggesting multiple roles for MUC16. In mouse serous epithelial ovarian cancer, MUC16 protein was detected at the apical surface of well-differentiated tumors, but not poorly differentiated tumors. These findings document the presence of MUC16 in murine ovarian cancer and in normal tissues and provide a foundation for future functional studies.

Novel mutations of the lipoprotein lipase gene associated with hypertriglyceridemia in members of type 2 diabetic pedigrees.

Increased plasma triglyceride and free fatty acid levels are frequently associated with type 2 diabetes mellitus (T2DM). To test the hypothesis that LPL gene mutations contribute to the hypertriglyceridemia observed in members of T2DM pedigrees, we screened the LPL gene in 53 hypertriglyceridemic members of 26 families. Four known and three novel mutations were identified. All three novel mutations, Lys312insC, Thr361insA, and double mutation Lys312insC + Asn291Ser, are clinically associated with hypertriglyceridemia. In vitro mutagenesis and expression studies confirm that these variants are associated with a significant reduction in LPL activity. The modeled structures displaying the Lys312insC and Thr361insA mutations showed loss of the activity-related C-terminal domain in the LPL protein. Another novel double mutation, Lys312insC + Asn291Ser, resulted in the loss of the catalytic ability of LPL attributable to the complete loss of the C-terminal domain and alteration in the heparin association site. Thus, these novel mutations of the LPL gene contribute to the hypertriglyceridemia observed in members of type 2 diabetic pedigrees.

Multiple histone deacetylases repress tumor suppressor gene ARHI in breast cancer.

ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast and ovarian epithelial cells but not in most breast and ovarian cancers. Our earlier studies showed that histone deacetylases (HDACs) in complexes with transcription factors E2F1 and E2F4 play an important role in downregulating ARHI expression in breast cancer cells. To determine which HDAC or HDACs are responsible for repressing ARHI, we cotransfected vectors expressing HDACs 1-11 with an ARHI/luciferase reporter into SKBr3 and MCF-7 breast cancer cells. Expression of multiple HDACs consistently reduced ARHI promoter activity in a dose-dependent manner. We also found that the expression level of HDACs 1-3 was higher in breast cancer cell lines than in normal breast epithelial cells. In agreement with their repressive function, depletion of HDACs 1, 3 and 11 not only significantly increased the ARHI promoter activity of the transfected reporter but also activated the transcription of the endogenous ARHI gene. Furthermore, depletion or inhibition of HDACs by small interfering RNA of HDAC11 or by trichostatin A, respectively, increased E2F acetylation. Chromatin immunoprecipitation assays revealed that HDACs 1 and 3 are bound to the ARHI promoter. Taken together, our results suggest that the activity of multiple HDACs contributes to the repression of the ARHI tumor suppressor gene in breast cancer cells. Since HDAC inhibitors are now being used to treat breast cancer, the reactivation of ARHI in these cancer cells may serve as a new biomarker with which to monitor the treatment effects.

Transcriptional and posttranscriptional down-regulation of the imprinted tumor suppressor gene ARHI (DRAS3) in ovarian cancer.

PURPOSE: ARHI expression is lost or markedly down-regulated in the majority of ovarian cancers. The mechanism by which ARHI is down-regulated in ovarian cancers is still not clear. Our previous reports indicated that ARHI promoter activity was reduced in ovarian cancer cells, due in part to the effects of negative regulatory transcription factor(s). EXPERIMENTAL DESIGN AND RESULTS: We now show that E2F1 and E2F4, but not E2F2, E2F3, or E2F5, bind to the ARHI promoter and repress its activity in ovarian cancer cells. Consistent with this observation, immunochemical staining of cell lines and of 364 samples of ovarian cancer tissue show that the expression of E2F1 and E2F4 proteins is much higher in ovarian cancer cells than in normal ovarian epithelial cells, and that increased expression of E2Fs was negatively correlated with ARHI expression (P < 0.05). Mutation of the putative E2F binding site in the ARHI promoter reversed this inhibitory effect and significantly increased ARHI promoter activity. In addition to the effects of transcriptional regulation, ARHI mRNA also exhibited a significantly reduced half-life in ovarian cancer cells when compared with that in normal ovarian epithelial cells (P < 0.01), suggesting posttranscriptional regulation of ARHI expression. ARHI mRNA contains AU-rich elements (ARE) in the 3'-untranslated region. We have found that these AREs interact with HuR, an ARE-binding protein that stabilizes bound mRNAs, possibly contributing to the rapid turnover of ARHI mRNA. Finally, reduced HuR ARE binding activity was observed in ovarian cancer cells when compared with normal ovarian surface epithelium. CONCLUSIONS: Taken together, our data suggest that ARHI expression is regulated at both the transcriptional and the posttranscriptional levels, contributing to the dramatic decrease in ARHI expression in ovarian cancers.

A Ras homologue member I directly inhibits signal transducers and activators of transcription 3 translocation and activity in human breast and ovarian cancer cells.

A Ras homologue member I (ARHI) is a novel imprinted tumor suppressor gene whose expression is frequently lost in breast and ovarian cancers. This small GTP-binding protein is a member of the Ras superfamily with significant homology to both Ras and Rap. Unlike the Ras oncogene, however, ARHI inhibits tumor cell growth. To elucidate the mechanisms by which ARHI inhibits cancer growth, we screened a human breast epithelial cell cDNA library using a yeast two-hybrid system for ARHI-interacting proteins. ARHI was found to interact with signal transducers and activators of transcription (STAT) 3, a latent transcription factor that transduces signals from the cell surface to the nucleus and activates gene transcription. STAT3 is frequently phosphorylated and activated in breast and ovarian cancers, where cytokines and growth factors up-regulate STAT3 and stimulate proliferation. The ARHI-STAT3 interaction was confirmed by coimmunoprecipitation in mammalian cells and shown to be specific for STAT3 but not STAT1 or STAT5a. When ARHI and STAT3 were coexpressed in SKOv3 cells, ARHI formed a complex with STAT3 in the cytoplasm and prevented interleukin-6-induced STAT3 accumulation in the nucleus. ARHI markedly reduced STAT3 binding to DNA and STAT3-dependent promoter activity while only moderately affecting STAT3 phosphorylation. Deletion of the NH2 terminus of ARHI significantly compromised its inhibitory activity, suggesting that this unique NH2-terminal extension contributes to ARHI's inhibition of STAT3-mediated transcriptional activity. Thus, the physical association between STAT3 and ARHI as well as the functional inhibition of STAT3 transcriptional activity by ARHI suggests a novel mechanism through which a putative tumor suppressor gene can inhibit STAT3 activity in breast and ovarian cancers.

Expression of the tumor suppressor gene ARHI in epithelial ovarian cancer is associated with increased expression of p21WAF1/CIP1 and prolonged progression-free survival.

PURPOSE: ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization. EXPERIMENTAL DESIGN: Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma. RESULTS: Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival. CONCLUSIONS: Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).

Loss of the expression of the tumor suppressor gene ARHI is associated with progression of breast cancer.

PURPOSE: Ductal carcinoma in situ (DCIS) is a preinvasive-stage breast carcinogenesis that accounts for approximately 20 approximately 25% of mammographically detected breast cancers. A significant fraction of untreated DCIS will evolve into invasive cancer. ras homologue I (ARHI) is an imprinted tumor suppressor gene that is expressed in normal breast epithelial cells but absent or down-regulated in breast cancer cells. This study investigated the relationship of ARHI expression to the progression of breast cancer. EXPERIMENTAL DESIGN: We analyzed ARHI expression in DCIS, invasive breast carcinoma, and adjacent normal breast epithelium from 64 formalin-fixed, paraffin-embedded DCIS specimens by both immunohistochemistry and in situ hybridization. We also analyzed the correlation between ARHI expression and progression of breast cancer, as well as the correlation of ARHI expression and cyclin D1 and p21(WAF1/CIP1) expression in DCIS. RESULTS: Normal breast epithelium was found in all of the specimens and invasive breast carcinoma was found in 23 specimens. ARHI mRNA and protein were detected in all of the normal breast epithelia. ARHI expression was detected mainly in cytoplasm and rarely present in the nucleus. By histochemical analysis, ARHI expression was down-regulated in 41% (26 of 64) of DCIS and 70% (16 of 23) of invasive carcinomas comparing the specimens with adjacent normal breast epithelium. When DCIS and invasive cancer were present in the same sample, ARHI was further down-regulated in 26% (6 of 23) of invasive carcinoma. In four cases [4 (17%) of 23] of invasive carcinoma, ARHI protein expression was totally lost. Consistent results were obtained with an in situ hybridization assay for ARHI at the mRNA level. Higher levels of expression of cyclin D1 and p21(WAF1/CIP1) were observed in DCIS than in the adjacent epithelia. The expression of cyclin D1 and p21(WAF1/CIP1) was inversely correlated with that of ARHI. CONCLUSIONS: Our results indicate that ARHI expression is markedly down-regulated in DCIS, and a further decrease in ARHI expression is associated with progression of breast cancer.

Aberrant methylation and silencing of ARHI, an imprinted tumor suppressor gene in which the function is lost in breast cancers.

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.

Reactivation of the silenced and imprinted alleles of ARHI is associated with increased histone H3 acetylation and decreased histone H3 lysine 9 methylation.

ARHI has been identified as a maternally imprinted tumor suppressor gene that maps to chromosome 1p31 and whose expression is markedly down-regulated in breast cancer. To explore possible mechanisms that could silence ARHI expression, we have tested the importance of DNA methylation, histone acetylation and histone methylation in regulating ARHI expression. We found that treatment with CpG demethylating agents and/or histone deacetylase inhibitors could reactivate both the silenced and the imprinted alleles of this tumor suppressor gene. Reactivation of ARHI expression by these reagents is related to the methylation status of the CpG islands in the ARHI promoter, especially CpG island II. Chromatin immunoprecipitation assays revealed that histone H3 lysine 9/18 acetylation levels associated with ARHI in normal cells were significantly higher than those in breast cancer cell lines that lacked ARHI expression. Treatment with a CpG demethylating agent and/or histone deacetylase inhibitor could increase ARHI expression in breast cancer cells, with a corresponding increase in histone H3 lysine 9/18 acetylation and decrease in histone H3 lysine 9 methylation.

ARHI is a Ras-related small G-protein with a novel N-terminal extension that inhibits growth of ovarian and breast cancers.

Our group recently identified Ras homolog member I (ARHI), a novel maternally imprinted tumor suppressor gene that encodes a 26 kDa GTP-binding protein with high homology to Ras and Rap. Unlike other Ras family members, ARHI exhibits several unusual structural and functional properties. ARHI contains a unique 34 amino-acid extension at the N-terminus, and differs from Ras in residues critical for GTPase activity and in its putative effector domain. Like Ras, ARHI can bind to GTP with high affinity but has low intrinsic GTPase activity. In addition, while Ras is an oncogene, ARHI functions as an inhibitor for cell growth. (32)Phosphorus labeling showed that ARHI is maintained in a constitutively activated GTP-bound state in resting cells, possibly because of impaired GTPase activity. ARHI is associated at the cell membrane through its prenylation at the C-terminal cysteine residue. Mutation of the conserved CAAX box at the C-terminus led to a loss of its membrane association and a decreased ability to inhibit cell growth. Conversion of Ser(51) to Asn decreased GTP binding and reduced ARHI's biological activity. Mutation of Ala(46) to Val increased the ability of ARHI to inhibit cell growth, associated with a further decrease of its intrinsic GTPase activity. Moreover, conversion of residues in ARHI that are conserved in the Ras family for GTPase activity partially restored the GTPase activity in ARHI. Most strikingly, deletion of ARHI's unique N-terminal extension nearly abolished its inhibitory effect on cell growth, suggesting its importance in ARHI's inhibitory function. Thus, ARHI is a unique Ras family member that retains basic small GTPase function, but exhibits many unusual features. In contrast to most other Ras family members, ARHI has a long N-terminal extension, modest GTPase activity, and constitutive GTP binding in resting cells. Furthermore, unlike the Ras oncogene, ARHI inhibits cell growth, and loss of its expression in cells may contribute to the development of breast and ovarian cancers.

Epigenetic regulation of ARHI in breast and ovarian cancer cells.

ARHI (Ras homologue member I) encodes a 26-kDa GTPase with 50-60% amino acid homology to Ras and Rap. ARHI and Ras share similar GTP/GDP binding domains, but exert opposite functions. ARHI is one of the first reported tumor suppressors in the ras superfamily. ARHI is expressed consistently in normal breast and ovarian epithelial cells, but not in breast or ovarian cancers. The loss of ARHI can be related to tumor progression. Reexpression of ARHI induces apoptosis of breast and ovarian cancer cells by a caspase-independent, calpain-dependent pathway. ARHI is consistently expressed in normal breast and ovarian epithelial cells but is dramatically downregulated in more then 70% of breast and ovarian cancers. ARHI is maternally imprinted with methylation of the three CpG islands in the maternal allele of normal cells. ARHI is expressed only from the paternal allele whose three CpG islands are not methylated. Loss of ARHI expression can occur through a genetic event, with loss of heterozygosity observed in 40% of breast, ovarian, and pancreatic cancers; but it can also occur through epigenetic mechanisms, including DNA methylation, histone deacetylation, histone methylation, and transcriptional regulation. Our data suggest that acetylation and methylation of chromatin associated with the ARHI promoter leads to loss of both ARHI expression and the ability to suppress tumor growth. Changes in chromatin that silence ARHI may be driven by methylation-dependent and -independent pathways. Reactivation of both the silenced paternal and imprinted maternal alleles can be achieved by demethylation and inhibition of histone deacetylation.