Inactivation of the Dlc1 gene cooperates with downregulation of p15INK4b and p16Ink4a, leading to neoplastic transformation and poor prognosis in human cancer.

The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEF) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15(Ink4b) and p16(Ink4a) together with increased expression and activation of Cdk4/6. The level of expression of these cell-cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (P < 0.01) with downregulation of p15(Ink4b) or p16(Ink4a) or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (P < 0.05) was associated with low DLC1 and low p15(Ink4b) in lung cancer and colon cancer, low DLC1 and low p16(Ink4a) in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.

Preclinical evaluation of combined antineoplastic effect of DLC1 tumor suppressor protein and suberoylanilide hydroxamic acid on prostate cancer cells.

Deleted in liver cancer (DLC1), a tumor suppressor gene in multiple cancers, is recurrently down regulated or inactivated by epigenetic mechanisms in primary prostate carcinomas (PCAs). In this study the methylation and acetylation profile of the DLC1 promoter region was examined in three PCA cell lines with low or undetectable DLC1 expression: LNCaP, its derivative C4-2B-2, and 22Rv1. Two histone deacetylase inhibitors (HDAC), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) induced histone acetylation of the DLC1 promoter in all three lines. DLC1 promoter methylation and deacetylation were detected in LNCaP and C4-2B-2 cells while in 22Rv1 cells DLC1 is silenced by deacetylation. Treatment with SAHA or TSA efficiently increased DLC1 expression in all lines, particularly in 22Rv1 cells, and activated the DLC1 promoter through the same Sp1 sites. The 22Rv1 cell line was selected to evaluate the efficacy of combined DLC1 transduction and SAHA treatment on tumor growth in athymic mice. Individually, DLC1 transduction and SAHA exposure reduced the tumor size by 75-80% compared to controls and in combination almost completely inhibited tumor growth. The antitumor effect was associated with the induction of apoptosis and inhibition of RhoA activity. SAHA alone significantly reduced RhoA activity, showing that this RhoGTPase is a target for SAHA. These results, obtained with a reliable preclinical in vivo test, predict that combined therapeutic agents targeting the pathways governing DLC1 function and HDAC inhibitors may be beneficial in management of prostate cancer.

Synergistic antineoplastic effect of DLC1 tumor suppressor protein and histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), on prostate and liver cancer cells: perspectives for therapeutics.

Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. The human tumor suppressor gene DLC1 (deleted in liver cancer 1) is frequently downregulated or silenced in multiple cancers, predominantly by epigenetic mechanisms. With the current considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly histone deacetylase (HDAC) inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction and exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor, in two human cancer cell lines that lack intrinsic DLC1 expression, 22Rv1 prostate cancer cells and 7703K human hepatocellular carcinoma cells. Consistent with the oncosuppressive function of DLC1 in several cancers, including prostate and liver cancer, transduction of 22Rv1 and 7703K cells with an Ad-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA (5 microM) efficiently restored the expression of DLC1 in 22Rv1 cells that lack DLC1 expression due to histone deacetylation but had a minimal effect in 7703K cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In 22Rv1 cells, this combination regimen nearly abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.

DLC1 suppresses distant dissemination of human hepatocellular carcinoma cells in nude mice through reduction of RhoA GTPase activity, actin cytoskeletal disruption and down-regulation of genes involved in metastasis.

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues.

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (approximately 2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (approximately 12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (approximately 0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharynx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.

Identification of genes related to nasopharyngeal carcinoma with the help of pathway-based networks.

cDNA microarray is a powerful tool to analyze simultaneously the expression levels of tens of thousands of genes. Compared with normal nasopharynx (NP) tissues, 2210 genes were highly differentially expressed in nasopharyngeal carcinoma (NPC) tissues detected by cDNA microarray. Since signal pathway is widely used to describe the complex relationship between genes, a pathway-based network was constructed to visualize the connection between the genes obtained from microarray data in this report. We analyzed the targeted genes that may have more important influence on this gene network with statistical methods and found that some genes might have significant influence on this network, especially Ras-related nuclear protein (RAN), carboxyl ester lipase (CEL), v-rel reticuloendotheliosis viral oncogene homolog A (RELA) genes. To verify the results from pathway-based selection, reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to detect the expression levels of RAN, CEL and RELA genes and it was found that the RAN and CEL genes were significantly up-regulated in more than 80% of NPC tissues. To further elucidate the function of the RAN gene, RAN expression was specifically suppressed in a 5-8F NPC cell line by RNA interference (RNAi). As expected, the depletion of RAN could effectively block the proliferation of tumor cells. Therefore, our study may open up a new way to analyze the vast microarray data.

Genetic and epigenetic alterations of DLC-1, a candidate tumor suppressor gene, in nasopharyngeal carcinoma.

The DLC-1 gene, located at the human chromosome region 8p22, behaves like a tumor suppressor gene and is frequently deleted in diverse tumors. The deletion of 8p22 is not an uncommon event in nasopharyngeal carcinoma (NPC), therefore we explored the expression levels of the DLC-1 gene in NPCs and NPC cell lines by reverse transcription-polymerase chain reaction. The results showed the mRNA level of DLC-1 was downregulated. To identify the mechanism of DLC-1 downregulation in NPC, we investigated the methylation status of the DLC-1 gene using methylation-specific PCR, and found that 79% (31 of 39) of the NPC tissues and two DLC-1 nonexpressing NPC cell lines, 6-10B and 5-8F, were methylated in the DLC-1 CpG island. Microsatellite PCR was also carried out, and loss of heterozygosity was found at four microsatellite sites (D8S552, D8S1754, D8S1790 and D8S549) covering the whole DLC-1 gene with ratios of 33% (4 of 12 informative cases), 18% (2 of 11), 5% (1 of 18), and 25% (3 of 12), respectively. Taken together, our results suggest that DLC-1 might be an NPC-related tumor suppressor gene affected by aberrant promoter methylation and gene deletion.

Identification of differentially expressed genes in metastatic and non-metastatic nasopharyngeal carcinoma cells by suppression subtractive hybridization.

BACKGROUND & OBJECTIVE: Nasopharyngeal carcinoma (NPC) is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. METHODS: Subtractive suppression hybridization (SSH) was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. RESULTS & DISCUSSION: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16), two were predicted genes (c9orf74 and MDS006), and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The remaining up-regulated genes identified in this study have not been reported to be markers of metastasis and may represent new candidates of NPC metastasis-related genes. The results of this study may provide novel points of therapeutic intervention for NPC.

Reexploring the possible roles of some genes associated with nasopharyngeal carcinoma using microarray-based detection.

In gene expression profiling, nasopharyngeal carcinoma (NPC) 5-8F cells differ from 6-10B cells in terms of their high tumorigenicity and metastatic ability. Differentially expressed genes from the two cell types were analyzed by combining with MILANO (the automatic custom annotation of microarray results which is based on all the available published work in PubMed). The results showed that five genes, including CTSD, P63, CSE1L, BPAG1 and EGR1, have been studied or mentioned in published work on NPC. Subsequently, we reevaluated the roles of these genes in the pathogenesis of NPC by combining the data of gene chips from NPCs versus NPs and pooled cells from 5-8F, 6-10B and CNE2 versus NPs. The results suggested that the roles of BPAG1 and EGR1 are possibly different from those reported in previous NPC studies. These five genes are likely to be involved in the proliferation, apoptosis, invasion and metastasis of NPC. A reexploration of the genes will further define their roles in the pathogenesis of NPC.

[Detection of RNA interference in nasopharyngeal carcinoma cell lines using reporter genes].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) technique is now widely used in studies of gene function, signal transduction pathway, and gene therapy because it can effectively and specifically inhibit gene expression. This study was designed to synthesize small interfering RNA (siRNA) by in vitro transcription, and construct retrovirus vectors to express small hairpin RNA (shRNA), detect RNAi in nasopharyngeal carcinoma cell lines, and to develop a RNAi technique platform. METHODS: siRNAs targeting green fluorescent protein (GFP) and luciferase (Luc) were synthesized by in vitro transcription, while shRNAs targeting GFP and Luc were constructed from pSUPER.retro. Cervical cancer cell line HeLa, nasopharyngeal carcinoma cell lines CNE1, CNE2, and 5-8F were co-transfected with siRNAs or shRNAs and reporter gene pEGFP-N1 or pGL3. The expression of GFP was detected by fluorescent microscopy and Western blot. The activity of luciferase was measured by Luciferase Enzyme Assay System. RESULTS: siRNA duplexes with 3' UU overhangs and shRNA specifically silenced GFP expression, while antisense RNA and siRNA without 3' UU overhangs did not trigger RNA interference of GFP. Quantitative luciferase activity analysis showed that siRNA inhibited Luc expression in HeLa, CNE1, CNE2, and 5-8F cell lines with inhibition rates of 91.43%, 78.01%, 90.30%, and 62.85%, respectively. Similarly, the inhibition rate was 78.22% when shRNA targeting Luc was co-transfected into HeLa cell line. CONCLUSIONS: Both siRNAs and shRNAs can induce RNAi. 3' UU overhangs of siRNA may play a role in RNAi. RNAi can be triggered in both nasopharyngeal carcinoma cell lines and HeLa cell line.