Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs.

Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

Loss of LKB1 in high-grade endometrial carcinoma: LKB1 is a novel transcriptional target of p53.

BACKGROUND: Liver kinase B1 (LKB1) is a serine/threonine kinase that functions as a tumor suppressor and regulates cell polarity, proliferation, and metabolism. Mutations in LKB1 are associated with Peutz-Jeghers syndrome as well as sporadic cervical and lung cancers. Although LKB1-null mice develop invasive endometrial cancers, the role and regulation of LKB1 in the pathogenesis of human endometrial cancer are not well defined and are the focus of these studies. METHODS: LKB1 protein and messenger RNA (mRNA) expression levels were evaluated in high-grade and low-grade endometrioid endometrial cancer (EEC) and cell lines by reverse transcriptase-polymerase chain reaction analysis, Western blot analysis, and immunohistochemistry. Mutational and promoter analyses of the LKB1 gene (serine/threonine kinase 11 [STK11]) were performed to identify the mechanisms that contribute to the loss of LKB1 in high-grade EEC. RESULTS: Analysis of the LKB1 gene in low-grade and high-grade EECs revealed no genetic mutations, suggesting that alterations in LKB1 transcription may be responsible for LKB1 protein loss in high-grade EEC. Analysis of the LKB1 promoter revealed 4 putative tumor protein 53 (p53) binding sites. Quantitative chromatin immunoprecipitation demonstrated that p53 bound directly to 1 of these sites and increased LKB1 promoter activity 140-fold. LKB1 promoter activity, mRNA, and protein levels were suppressed after silencing of p53 with small interfering RNA and were elevated in cells that overexpressed p53. Levels of p53 mRNA and protein expression were decreased in high-grade EEC and were positively correlated with LKB1 protein levels (Spearman correlation, r=0.601; P<.001). CONCLUSIONS: LKB1 is a direct transcriptional target of p53. The loss of wild-type p53 in high-grade EEC may contribute to the LKB1 loss observed in these more aggressive tumors.

PFTK1 interacts with cyclin Y to activate non-canonical Wnt signaling in hepatocellular carcinoma.

PFTK1 is a Cdc2-related protein kinase that is frequently upregulated in human hepatocellular carcinoma (HCC) where it correlates with metastatic features and motile phenotypes. To understand the modulated pathway underlining the PFTK1 action, here we show a physical interaction between PFTK1 and cyclin Y (CCNY) in promoting noncanonical Wnt signaling. In HCC cells, we found PFTK1 forms a direct complex with CCNY, and together readily upregulate key components of Wnt signaling (Dvl2 and Naked1). Exogenous expression of PFTK1 and CCNY activated Rho GTPases, which are known targets of the noncanonical path. In line with Rho GTPases activation, we also found marked actin polymerizations in cells with PFTK1-CCNY co-expressions. Our findings highlight a PFTK1-CCNY complex in activating noncanonical Wnt signaling in HCC cells.

ViralFusionSeq: accurately discover viral integration events and reconstruct fusion transcripts at single-base resolution.

SUMMARY: Insertional mutagenesis from virus infection is an important pathogenic risk for the development of cancer. Despite the advent of high-throughput sequencing, discovery of viral integration sites and expressed viral fusion events are still limited. Here, we present ViralFusionSeq (VFS), which combines soft-clipping information, read-pair analysis and targeted de novo assembly to discover and annotate viral-human fusions. VFS was used in an RNA-Seq experiment, simulated DNA-Seq experiment and re-analysis of published DNA-Seq datasets. Our experiments demonstrated that VFS is both sensitive and highly accurate. AVAILABILITY: VFS is distributed under GPL version 3 at http://hkbic.cuhk.edu.hk/software/viralfusionseq

Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma.

BACKGROUND & AIMS: Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. METHODS: Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. RESULTS: The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients (p<0.009). CONCLUSIONS: Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.

P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells.

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.

Understanding obesity and endometrial cancer risk: opportunities for prevention.

Worldwide, obesity has become a major public health crisis. Overweight and obesity not only increase the risk of cardiovascular disease and type-2 diabetes mellitus but also are now known risk factors for a variety of cancer types. Among all cancers, increasing body mass index is associated most strongly with endometrial cancer incidence and death. The molecular mechanisms underlying how adipose tissue and obesity contribute to the pathogenesis of endometrial cancer are becoming better understood and have revealed a number of rational strategies, both behavioral and pharmaceutical, for the prevention of both primary and recurrent disease.

ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

Mechanistic study on growth suppression and apoptosis induction by targeting hepatoma-derived growth factor in human hepatocellular carcinoma HepG2 cells.

Hepatoma-derived growth factor (HDGF) is frequently overexpressed in human cancer. The growth factor was previously demonstrated to be a survival factor as knock-down of HDGF suppresses the growth and induces apoptosis in human cancer cells through the Bad-mediated intrinsic apoptotic pathway. However, inactivation of Bad cannot completely repress the apoptosis induced upon HDGF knock-down, indicating the presence of other unidentified pathways. In the present study, HDGF knock-down was shown to trigger the Fas-mediated extrinsic apoptotic pathway in human hepatocellular carcinoma HepG2 cells through NF-kappaB signaling pathway. Increases in Fas expression and fas promoter activity were detected upon HDGF knock-down by Western blot analysis and luciferase reporter assay. Knock-down of fas inhibited HDGF knock-down effect on apoptosis induction and growth suppression as revealed by annexin V binding assay and soft agar assay. Down-regulation of IkappaBalpha was also observed upon HDGF knock-down. Overexpression of IkappaBalpha by transient transfection or inhibition of NF-kappaB by BAY11-7082 suppressed HDGF knock-down effect on fas promoter activation, Fas up-regulation, apoptosis induction and growth suppression. Furthermore, the interaction of Fas-mediated extrinsic and Bad-mediated intrinsic apoptotic pathways was demonstrated as a stronger inhibition on apoptosis induction and growth suppression upon HDGF knock-down was observed when both pathways were inactivated. The results therefore suggested that, through both intrinsic and extrinsic apoptotic pathways, HDGF may function as a survival factor and be a potential target for cancer therapy.

Facilitation of drug resistance development by gamma-irradiation in human cancer cells.

Hepatocellular carcinoma HepG2 cells (G cells) were subjected to selection first with gamma-radiation and then doxorubicin (Dox). The radiation treatment consisted of 2 Gy for 10 days (G2) or 10 Gy for 2 days (G10) and the Dox treatment was continuous exposure for up to 10 microM. Compared with respective parental G, G2, G10 cells, the Dox-selected cells showed mdr1 amplification/P-glycoprotein overexpression, Dox resistance and also less intracellular Dox accumulation. Verapamil reversed the drug resistance and increased the Dox accumulation in all cells. Decay in drug resistance and reduction in mdr1 amplification/P-glycoprotein overexpression were observed in the Dox-selected cells culturing in Dox-free condition. Among the Dox-selected cells, G2R cells showed the highest levels of drug resistance, mdr1 amplification, but the least resistance decay. Results from the study indicate the possible influence of radiation treatment on the development of drug resistance in cancer cells and it may even lead to a highly resistant phenotype.

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway.

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, gamma-radiation, IFN-alpha, and IFN-gamma in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance.

Downregulation of hepatoma-derived growth factor activates the Bad-mediated apoptotic pathway in human cancer cells.

Hepatoma-derived growth factor (HDGF) is highly expressed in human cancer and its expression is correlated with poor prognosis of cancer. The growth factor is known to stimulate cell growth while the underlying mechanism is however not clear. Transfection with HDGF cDNA stimulated while its specific antisense oligonucleotides repressed the growth of human hepatocellular carcinoma HepG2 cells. Furthermore, knock-down of HDGF by antisense oligos also induced apoptosis in HepG2 cells and in other human cancer cells, e.g. human squamous carcinoma A431 cells. HDGF knock-down was found to induce the expression of the pro-apoptotic protein Bad and also inactivate ERK and Akt, which in turn led to dephosphorylation of Bad at Ser-112, Ser-136, and activation of the intrinsic apoptotic pathway, i.e. depolarization of the mitochondrial membrane, release of mitochondrial cytochrome c, increase in the processing of caspase 9 and 3. As HDGF knock-down not only suppresses the growth but also induces apoptosis in human cancer cells, HDGF may therefore serve as a survival factor for human cancer cells and a potential target for cancer therapy.

p53-R273H gains new function in induction of drug resistance through down-regulation of procaspase-3.

Development of drug resistance is one of the major obstacles in cancer chemotherapy. The molecular mechanism leading to drug resistance is still not fully understood. A10A cells, a doxorubicin-resistant subline of human squamous cell carcinoma A431 cells, showed cross-resistance to methotrexate and also resistance to the drug-induced apoptosis. The cells also showed overexpression of a mutated form of p53, p53-R273H (Arg to His at codon 273), and down-regulation of procaspase-3. Knockdown of p53-R273H by p53 small interfering RNA in A431 cells increased procaspase-3 level and sensitized the cells to drug-induced apoptosis. On the other hand, transfection of p53-R273H into p53 null human osteosarcoma Saos-2 cells down-regulated procaspase-3 level and induced resistance to the drug toxicity and drug-induced apoptosis. The results support the idea that p53-R273H may gain new functions in induction of drug resistance and impairment in drug-induced apoptosis through down-regulation of procaspase-3 level. The study sheds new light on the understanding of the gain of function and drug resistance mechanisms associated with mutant p53.

Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1.

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

Viral-human chimeric transcript predisposes risk to liver cancer development and progression.

The mutagenic effect of hepatitis B (HBV) integration in predisposing risk to hepatocellular carcinoma (HCC) remains elusive. In this study, we performed transcriptome sequencing of HBV-positive HCC cell lines and showed transcription of viral-human gene fusions from the site of genome integrations. We discovered tumor-promoting properties of a chimeric HBx-LINE1 that, intriguingly, functions as a hybrid RNA. HBx-LINE1 can be detected in 23.3% of HBV-associated HCC tumors and correlates with poorer patient survival. HBx-LINE1 transgenic mice showed heightened susceptibility to diethylnitrosamine-induced tumor formation. We further show that HBx-LINE1 expression affects ß-catenin transactivity, which underlines a role in activating Wnt signaling. Thus, this study identifies a viral-human chimeric fusion transcript that functions like a long noncoding RNA to promote HCC.

Another surprise from Metformin: novel mechanism of action via K-Ras influences endometrial cancer response to therapy.

Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K-targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin.

Identification of FGFR4 as a potential therapeutic target for advanced-stage, high-grade serous ovarian cancer.

PURPOSE: To evaluate the prognostic value of fibroblast growth factor receptor 4 (FGFR4) protein expression in patients with advanced-stage, high-grade serous ovarian cancer, delineate the functional role of FGFR4 in ovarian cancer progression, and evaluate the feasibility of targeting FGFR4 in serous ovarian cancer treatment. EXPERIMENTAL DESIGN: Immunolocalization of FGFR4 was conducted on 183 ovarian tumor samples. The collected FGFR4 expression data were correlated with overall survival using Kaplan-Meier and Cox regression analyses. The effects of FGFR4 silencing on ovarian cancer cell growth, survival, invasiveness, apoptosis, and FGF1-mediated signaling pathway activation were evaluated by transfecting cells with FGFR4-specific siRNAs. An orthotopic mouse model was used to evaluate the effect of injection of FGFR4-specific siRNAs and FGFR4 trap protein encapsulated in nanoliposomes on ovarian tumor growth in vivo. RESULTS: Overexpression of FGFR4 protein was significantly associated with decreased overall survival durations. FGFR4 silencing significantly decreased the proliferation, survival, and invasiveness and increased apoptosis of ovarian cancer cells. Also, downregulation of FGFR4 significantly abrogated the mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB), and WNT signaling pathways, which are activated by FGF1. Targeting FGFR4 with the FGFR4-specific siRNAs and FGFR4 trap protein significantly decreased ovarian tumor growth in vivo. CONCLUSIONS: FGFR4 is a prognostic marker for advanced-stage, high-grade serous ovarian carcinoma. Silencing FGFR4 and inhibiting ligand-receptor binding significantly decrease ovarian tumor growth both in vitro and in vivo, suggesting that targeting ovarian cancer cells with high levels of FGFR4 protein expression is a new therapeutic modality for this disease and will improve survival of it.

Clitocine reversal of P-glycoprotein associated multi-drug resistance through down-regulation of transcription factor NF-κB in R-HepG2 cell line.

Multidrug resistance (MDR) is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.

HPV-16 E6 upregulation of DNMT1 through repression of tumor suppressor p53.

The HPV-16 early proteins E6 and E7 are considered to function as oncoproteins in cervical cancer. DNA methyltransferase 1 (DNMT1) is one of the enzymes involved in epigenetic silencing of tumor suppressor genes. In the present study, the functional role and regulation of DNMT1 in HPV-16 E6 associated cervical cancer development were examined. Knockdown of E6 in HPV-16 positive human cervical carcinoma SiHa and CaSki cells led to the increase in p53, repression of DNMT1 protein and promoter activity. Moreover, p53 knockdown increased the DNMT1 protein as well as promoter activity, indicating that p53 may mediate E6 upregulation of DNMT1. In addition, E6 knockdown induced growth retardation in SiHa cells, and the effect was partially reverted by DNMT1 overexpression. The results suggest that HPV-16 E6 may act through p53/ DNMT1 to regulate the development of cervical cancer.

AF1q enhancement of gamma irradiation-induced apoptosis by up-regulation of BAD expression via NF-kappaB in human squamous carcinoma A431 cells.

BAD (BCL-2 antagonist of cell death) is a pro-apoptotic BCL-2 family protein that plays a critical role in the regulation of apoptotic response. This study presents direct evidence that AF1q increased the radiation-induced apoptosis through up-regulation of BAD in human squamous carcinoma A431 cells and the key transcription factor involved is NF-kappaB. The minimal promoter sequence of BAD was identified; the activity was increased in AF1q stable transfectants and decreased upon AF1q siRNA transfection. The NF-kappaB consensus binding sequence is detected on BAD promoter. Inactivation of NF-kappaB by NF-kappaB inhibitor Bay 11-7082 or NF-kappaB p65 siRNA suppressed the expression and promoter activity of BAD; the suppression is more obvious in AF1q stable transfectants which also have an elevated NF-kappaB level. Mutation of putative NF-kappaB motif decreased the BAD promoter activity. The binding of NF-kappaB to the BAD promoter was confirmed by chromatin-immunoprecipitation. These findings indicate that AF1q up-regulation of BAD is through its effect on NF-kappaB and this may hint of its oncogenic mechanism in cancer.