Jack Wu

Expression profile of microRNAs in c-Myc induced mouse mammary tumors

Abstractc-Myc is a transcription factor overexpression of which induces mammary cancer in transgenic mice. To explore whether certain microRNAs (mirRNA) mediate c-Myc induced mammary carcinogenesis, we studied mirRNA expression profile in mammary tumors developed from MMTV-c-myc transgenic mice, and found 50 and 59 mirRNAs showing increased and decreased expression, respectively, compared with lactating mammary glands of wild type mice. Twenty-four of these mirRNAs could be grouped into eight clusters because they had the same chromosomal localizations and might be processed from the same primary RNA transcripts. The increased expression of mir-20a, mir-20b, and mir-9 as well as decreased expression of mir-222 were verified by RT-PCR, real-time RT-PCR, and cDNA sequencing. Moreover, we fortuitously identified a novel non-coding RNA, the level of which was decreased in proliferating mammary glands of MMTV-c-myc mice was further decreased to undetectable level in the mammary tumors. Sequencing of this novel RNA revealed that it was transcribed from a region of mouse chromosome 19 that harbored the metastasis associated lung adenocarcinoma transcript-1 (Malat-1), a non-protein-coding gene. These results suggest that certain mirRNAs and the chromosome 19 derived non-coding RNAs may mediate c-myc induced mammary carcinogenesis.

An MDM2 antagonist (MI-319) restores p53 functions and increases the life span of orally treated follicular lymphoma bearing animals

BackgroundMI-319 is a synthetic small molecule designed to target the MDM2-P53 interaction. It is closely related to MDM2 antagonists MI-219 and Nutlin-3 in terms of the expected working mechanisms. The purpose of this study was to evaluate anti-lymphoma activity of MI-319 in WSU-FSCCL, a B-cell follicular lymphoma line. For comparison purpose, MI-319, MI-219 and Nutlin-3 were assessed side by side against FSCCL and three other B-cell hematological tumor cell lines in growth inhibition and gene expression profiling experiments.ResultsMI-319 was shown to bind to MDM2 protein with an affinity slightly higher than that of MI-219 and Nutlin-3. Nevertheless, cell growth inhibition and gene expression profiling experiments revealed that the three compounds have quite similar potency against the tumor cell lines tested in this study. In vitro, MI-319 exhibited the strongest anti-proliferation activity against FSCCL and four patient cells, which all have wild-type p53. Data obtained from Western blotting, cell cycle and apoptosis analysis experiments indicated that FSCCL exhibited strong cell cycle arrest and significant apoptotic cell death; cells with mutant p53 did not show significant apoptotic cell death with drug concentrations up to 10 μM, but displayed weaker and differential cell cycle responses. In our systemic mouse model for FSCCL, MI-319 was tolerated well by the animals, displayed effectiveness against FSCCL-lymphoma cells in blood, brain and bone marrow, and achieved significant therapeutic impact (p < 0.0001) by conferring the treatment group a > 28% (%ILS, 14.4 days) increase in median survival days.ConclusionOverall, MI-319 probably has an anti-lymphoma potency equal to that of MI-219 and Nutlin-3. It is a potent agent against FSCCL in vitro and in vivo and holds the promises to be developed further for the treatment of follicular lymphoma that retains wild-type p53.

Identification and functional analysis of 9p24 amplified genes in human breast cancer

Previously, our group identified a novel amplicon at chromosome 9p24 in human esophageal and breast cancers, and cloned the novel gene, GASC1 (gene amplified in squamous cell carcinoma 1, also known as JMJD2C/KDM4C), from this amplicon. GASC1 is a histone demethylase involved in the deregulation of histone methylation in cancer cells. In the current study, we aimed to comprehensively characterize the genes in the 9p24 amplicon in human breast cancer. We performed extensive genomic analyses on a panel of cancer cell lines and narrowed the shortest region of overlap to approximately 2 Mb. Based on statistical analysis of copy number increase and overexpression, the 9p24 amplicon contains six candidate oncogenes. Among these, four genes (GASC1 UHRF2, KIAA1432 and C9orf123) are overexpressed only in the context of gene amplification while two genes (ERMP1 and IL33) are overexpressed independent of the copy number increase. We then focused our studies on the UHRF2 gene, which has a potential involvement in both DNA methylation and histone modification. Knocking down UHRF2 expression inhibited the growth of breast cancer cells specifically with 9p24 amplification. Conversely, ectopic overexpression of UHRF2 in non-tumorigenic MCF10A cells promoted cell proliferation. Furthermore, we demonstrated that UHRF2 has the ability to suppress the expression of key cell-cycle inhibitors, such as p16INK4a, p21Waf1/Cip1 and p27Kip1. Taken together, our studies support the notion that the 9p24 amplicon contains multiple oncogenes that may integrate genetic and epigenetic codes and have important roles in human tumorigenesis.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer.

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes. (Mol Cancer Res 2007;5(2):171–81)

MDM2 inhibitor MI-319 in combination with cisplatin is an effective treatment for pancreatic cancer independent of p53 function

Small molecule inhibitors (SMIs) of murine double minute 2 (MDM2) are known to restore the apoptotic and cell cycle regulatory functions of p53 by disrupting the MDM2-p53 interaction. In principle, these SMIs are not effective against tumours with mutation in the tumour suppressor p53 (mut-p53), which is known to be present in approximately 50% of all cancers. In this study we are reporting, for the first time, that MI-319 in combination with cisplatin induced cell growth inhibition and apoptosis in pancreatic cancer (PC) cells irrespective of their p53 mutational status. MI-319-cisplatin combination synergistically suppressed cell growth (MTT Combination Index [CI]<1) and colony formation (clonogenic assay) and induced apoptosis. Western blot analysis and siRNA silencing studies in mutant as well as p53 null cells highlighted a mechanism involving p73 which is also known to be under the regulation of MDM2, and unlike p53, it is rarely mutated in PC. Down-regulating MDM2 using siRNA enhanced p73 reactivation and increased cell death. Further, the combination effectively reduced tumour growth in both wt-p53 and mut-p53 tumour xenograft models (50% Capan-2 animals were tumour free). Consistent with our in vitro results, remnant tumour tissue analysis showed up-regulation of p73 and the cell cycle regulator p21. In conclusion, this study highlights a new role of MDM2 inhibitors in combination with cisplatin, and thus warrants further clinical investigation in human pancreatic tumours containing both wt-p53 and mut-p53.

Anti-invasive and antimetastatic activities of ribosomal protein S6 kinase 4 in breast cancer cells

Purpose: We have previously shown that p90 ribosomal protein S6 kinase 4 (RSK4), an X-linked gene, is highly up-regulated in mammary tumors of MMTV-c-Myc transgenic mice. In this study, we further investigated whether RSK4 inhibits or promotes breast tumor growth and progression. Experimental Design: Stable overexpression or small interfering RNA–mediated knockdown of RSK4 was done in the MDA-MB-231 cell line. Stable clones were tested for cell proliferation, anchorage-independent growth in soft agar, invasive and metastatic ability of these clones in vitro and tumorigenesis, invasive and metastatic ability in vivo in severe combined immunodeficient mice. Results: Here, we show that exogenous expression of RSK4 resulted in decreased cell proliferation and increased accumulation of cells in G0-G1 phase, which paralleled with enhanced expression of tumor suppressor genes: retinoblastoma protein, retinobl astoma-associated 46 kDa protein, and p21 protein. Overexpression of RSK4 resulted in reduced colony formation in soft agar and suppressed invasive and migratory activities of MDA-MB-231 cells both in vitro and in vivo. Importantly, RSK4-overexpressing cells showed up-regulation of claudin-2 and down-regulation of CXCR4, both of these play roles in invasion and chemotaxis. Conclusions: These results indicate that RSK4 expression may limit the oncogenic, invasive, and metastatic potential of breast cancer cells. Anti-invasive and antimetastatic activities of RSK4 may be, in part, due to its regulation of claudin-2. Increased expression of RSK4 in c-Myc-overexpressing cells and a dose-dependent induction of luciferase reporter gene activity suggest that c-Myc may regulate RSK4 expression.

Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo

Apogossypolone (ApoG2) is a semi-synthesized derivative of gossypol. The principal objective of this study was to compare stability and toxicity between ApoG2 and gossypol, and to evaluate anti-lymphoma activity of ApoG2 in vitro and in vivo. ApoG2 shows better stability when compared with a racemic gossypol and can be better tolerated by mice compared to gossypol. ApoG2 showed significant inhibition of cell proliferation of WSU-DLCL2 and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC50 of ApoG2 was determined to be 350 nM against WSU-DLCL2 cells. Treatment with ApoG2 at 600 mg/kg resulted in significant growth inhibition of WSU-DLCL2 xenografts. When combined with CHOP, ApoG2 displayed even more complete inhibition of tumor growth. ApoG2 binds to purified recombinant Bcl-2, Mcl-1, and Bcl-XL proteins with high affinity and is shown to block the formation of heterodimers between Bcl-XL and Bim. For a treatment of 72 h, ApoG2 induced a maximum of 32% of apoptotic cell death. Western blot experiments showed that treatment with ApoG2 led to cleavage of caspase-3, caspase-9 and PARP. Moreover, pretreatment of DLCL2 cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL2 cells at least partly by inducing apoptosis. It is an attractive small molecule inhibitor of the Bcl-2 family proteins to be developed further for the treatment of diffuse large cell lymphoma.

Synergistic Effect of Cyclin D1 and c-Myc Leads to More Aggressive and Invasive Mammary Tumors in Severe Combined Immunodeficient Mice

Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer; yet, it is not clear whether cyclin D1 alone is capable of causing malignant transformation of mammary epithelial cells. Here, we show that ectopic expression of cyclin D1 in benign mouse mammary epithelial cells promotes cell proliferation, anchorage-independent growth in soft agar, and tumorigenesis in severe combined immunodeficient mice. To address the possible interaction of cyclin D1 and c-myc in malignant transformation, we used cyclin D1/c-myc dual-expressing clones, which displayed more aggressive and invasive phenotype than cyclin D1-expressing clones. These data provide evidence that overexpression of cyclin D1 or coexpression with c-myc could cause invasive malignant transformation of benign mouse mammary epithelial cells. Furthermore, microarray analysis of cyclin D1 and cyclin D1/c-myc clones showed that these two tumor-producing clones might use distinct invasive pathways. In summary, overexpression of cyclin D1 may commit mammary epithelia to a tumor-prone phenotype in which cooperation with other genes, such as synergy with c-myc, may lead to a more aggressive phenotype.

Snail nuclear transport: The gateways regulating epithelial-to-mesenchymal transition?

Epithelial-to-mesenchymal transition (EMT) and the reverse process (MET) play central role in organ developmental biology. It is a fine tuned process that when disturbed leads to pathological conditions especially cancers with aggressive and metastatic behavior. Snail is an oncogene that has been well established to be a promoter of EMT through direct repression of epithelial morphology promoter E-cadherin. It can function in the nucleus, in the cytosol and as discovered recently, extracellularly through secretory vesicular structures. The intracellular transport of snail has for long been shown to be regulated by the nuclear pore complex. One of the Karyopherins, importin alpha, mediates snail import, while exportin 1 (Xpo1) also known as chromosome maintenance region 1 (CRM1) is its major nuclear exporter. A number of additional biological regulators are emerging that directly modulate Snail stability by altering its subcellular localization. These observations indicate that targeting the nuclear transport machinery could be an important and as of yet, unexplored avenue for therapeutic intervention against the EMT processes in cancer. In parallel, a number of novel agents that disrupt nuclear transport have recently been discovered and are being explored for their anti-cancer effects in the early clinical settings. Through this review we provide insights on the mechanisms regulating snail subcellular localization and how this impacts EMT. We discuss strategies on how the nuclear transport function can be harnessed to rein in EMT through modulation of snail signaling.

Targeting the Nuclear Export Protein XPO1/CRM1 Reverses Epithelial to Mesenchymal Transition.

Here we demonstrate for the first time that targeted inhibition of nuclear exporter protein exportin 1 (XPO1) also known as chromosome maintenance region 1 (CRM1) by Selective Inhibitor of Nuclear Export (SINE) compounds results in reversal of EMT in snail-transduced primary human mammary epithelial cells (HMECs). SINE compounds selinexor (KPT-330) and KPT-185, leptomycin B (LMB as +ve control) but not KPT-301 (–ve control) reverse EMT, suppress mesenchymal markers and consequently induce growth inhibition, apoptosis and prevent spheroid formation. SINE treatment resulted in nuclear retention of snail regulator FBXL5 that was concurrent with suppression of snail and down-regulation of mesenchymal markers. FBXL5 siRNA or transfection with cys528 mut-Xpo1 (lacking SINE binding site) markedly abrogated SINE activity highlighting an XPO1 and FBXL5 mediated mechanism of action. Silencing XPO1 or snail caused re-expression of FBXL5 as well as EMT reversal. Pathway analysis on SINE treated HMECs further verified the involvement of additional F-Box family proteins and confirmed the suppression of snail network. Oral administration of selinexor (15 mg/kg p.o. QoDx3/week for 3weeks) resulted in complete cures (no tumor rebound at 120 days) of HMLER-Snail xenografts. These findings raise the unique possibility of blocking EMT at the nuclear pore.