Abstract Background: In spite of the established role of oncogenic Kras in the development and subsistence of Pancreatic Cancer (PC), no clinically viable agents have been developed to suppress this master regulator. Unlike other druggable proteins, Kras lacks the ideal binding pocket for small molecules. Therefore, there is an unmet need to identify novel druggable sites on Kras or to develop agents that target key effector proteins downstream. The p21-activated kinase 4 (PAK4) is a key effector downstream of Rho family GTPases. PAK4 is over-expressed in most PC cell lines tested but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts confirmed PAK4 amplification. RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target. In collaboration with Karyopharm Therapeutics, we developed novel PAK4 allosteric modulators (PAMs; KPT-7189, KPT-9274, KPT-9307). Methods: Using multiple molecular biology techniques we tested PAMs activity (in the presence and absence of -ve and +ve controls) on a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted cancer stem cells (CSC). Pancreatic CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of PAMs were evaluated in vitro and in sub-cutaneous mouse models of PC. Results: The novel, orally bioavailable PAMs show anti-proliferative activity in vitro against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s <250nM) while sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in 5 different PC cell lines (not in HPDE). PAMs reduced RNA and PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). Confocal, western blot and RT-PCR analysis of PAM-treated CSC spheroids showed reversal of EMT and suppression of stem markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. Additionally PAMs synergize with Gemcitabine and oxaliplatin (CI<1) in vitro. KPT-9274, possesses desirable PK properties and is well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy (as a single agent and in combination with gemcitabine) in a sub-cutaneous, orthotopic (from primary cells) and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. Conclusions: These proof of concept studies demonstrating the anti-proliferative effects of novel PAK4 allosteric modulators in pancreatic cancer warrant further clinical investigations. Citation Format: Asfar S. Azmi, William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Sharon Shacham, Jack Wu, Amro Aboukameel, Irfana Muqbil, Ramzi M. Mohammad. Overcoming drug resistance and stemness in oncogenic kras driven pancreatic ductal adenocarcinoma through PAK4 inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4688. doi:10.1158/1538-7445.AM2015-4688

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.

The histone lysine demethylase KDM4 subfamily, comprised of four members (A, B, C, and D), play critical roles in controlling transcription, chromatin architecture and cellular differentiation. We previously demonstrated that KDM4C is significantly amplified and overexpressed in aggressive basal-like breast cancers and functions as a transforming oncogene. However, information regarding the genomic and transcriptomic alterations of the KDM4 subfamily in different subtypes of breast cancer remains largely incomplete. Here, we conducted a meta-analysis of KDM4A, B, C and D in breast cancer and identified associations among recurrent copy number alterations, gene expression and breast cancer subtypes. We demonstrated that KDM4A and D are also significantly overexpressed in basal-like breast cancer, whereas KDM4B overexpression is more dominant in estrogen-receptor-positive, luminal breast cancer. Next, we investigated the therapeutic potential of a novel histone demethylase inhibitor, NCDM-32B, in breast cancer. The treatment of basal breast cancer cell lines with NCDM-32B resulted in the decrease of cell viability and anchorage independent growth in soft agar. Furthermore, we found that NCDM-32B impaired several critical pathways that drive cellular proliferation and transformation in breast cancer. Our findings demonstrate genetic amplification and overexpression of the KDM4 demethylases in different subtypes of breast cancer. Furthermore, histone methylation is reversible and KDM4 demethylases are druggable targets. Thus, KDM4 inhibitors may serve as a novel therapeutic approach for a subset of aggressive breast cancer.

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.

Abstract Introduction: In spite of the well-recognized role of oncogenic K-Ras in the establishment and sustenance of the highly fatal disease, pancreatic cancer (PC), development of clinical agents that can tame this master regulator have been unsuccessful. This is primarily because, unlike other druggable targets, K-Ras lacks an ideal binding pocket that can be used to design small molecule drugs. Therefore, there is an urgent need to identify novel druggable sites in K-Ras or to develop agents that can target key effector proteins downstream of K-Ras signaling. To this end, we are pursuing p21-activated kinase 4 (PAK4) as a novel target for PC. PAK4 acts as a key effector of Rho family GTPases downstream of K-Ras and is found over-expressed in most of the available PC cell lines but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts has shown amplification of PAK4. Most importantly, RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target within the K-Ras signaling network. Nevertheless, the previously developed PAK4 Type I ATP competitive inhibitor (PF-3758309; tested in non-pancreatic models) was evaluated in a Phase 1 study and showed undesirable pharmacokinetic properties as well as no objective responses and was subsequently discontinued. In order to fill this scientific void, we evaluated a new class of PAK4 allosteric modulators in pancreatic cancer models. Experimental Procedure: Using multiple molecular biology techniques we tested the Pak4 modulators' activities (in the presence and absence of -ve and +ve controls) in a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted PC stem cells (CSC). CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of these PAK4 modulators were evaluated in sub-cutaneous mouse models of PC. Results: The novel, orally available PAK4 allosteric modulators (KPT-7189, KPT-8752) show anti-proliferative activity against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s <250nM) sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony forming abilities in 5 different PC cell lines (not in HPDE). KPT-7189 reduced PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Molecularly, PAK4 RNA interference enhanced KPT-7189 activity and co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). KPT-7189 inhibited spheroid forming ability of CSCs and reversed the epithelial-to-mesenchymal (EMT) phenotype. Confocal, western blot and RT-PCR analysis of KPT-7189 treated CD33+CD44+EpCam+ spheroids showed suppression of EMT and CSC markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. A similar compound, KPT-8752, with desirable PK properties was well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy trial in sub-cutaneous, orthotopic (developed from human primary cells) and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. Conclusion: This is the first proof of concept study demonstrating the anti-proliferative effects of novel allosteric modulators of PAK4, a downstream effector of K-Ras, in pancreatic cancer that warrants further clinical investigations. Citation Format: ASFAR S. AZMI, William T. Senapedis, Yosef Landesman, Erkan Baloglu, Bin Bao, Jack Wu, Kalid Ori, Sharon Shacham, Michael Kauffman, Ramzi M. Mohammad. Novel small molecule PAK4 allosteric modulators show potency against oncogenic K-Ras driven pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A24. doi: 10.1158/1557-3125.RASONC14-A24

Abstract The p21-activated kinase 4 (PAK4) acts as a key effector of Rho family GTPases downstream of K-Ras and is found over-expressed in most of the available pancreatic cancer (PC) cell lines but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts has shown amplification of PAK4. Most importantly, RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target within the K-Ras signaling network. Nevertheless, the previously developed PAK4 Type I ATP competitive inhibitor (PF-3758309; tested in non-pancreatic models) was evaluated in a Phase 1 study and showed undesirable pharmacokinetic properties as well as no objective responses and was subsequently discontinued. In order to fill this scientific void, we evaluated a new class of PAK4 allosteric modulators in PC. Using multiple molecular biology techniques we tested the Pak4 modulators' activities (in the presence and absence of -ve and +ve controls) in a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted PC stem cells (CSC). CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of these PAK4 modulators were evaluated in sub-cutaneous mouse models of PC. The novel, orally available PAK4 allosteric modulators (KPT-7189, KPT-8752) show anti-proliferative activity against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s <250nM) sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony forming abilities in 5 different PC cell lines (not in HPDE). KPT-7189 reduced PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Molecularly, PAK4 RNA interference enhanced KPT-7189 activity and co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). KPT-7189 inhibited spheroid forming ability of CSCs and reversed the epithelial-to-mesenchymal (EMT) phenotype. Confocal, western blot and RT-PCR analysis of KPT-7189 treated CD33+CD44+EpCam+ spheroids showed suppression of EMT and CSC markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. A similar compound, KPT-8752, with desirable PK properties was well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy trial in sub-cutaneous, orthotopic and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing. This is the first proof of concept study demonstrating the anti-proliferative effects of novel allosteric modulators of PAK4, a downstream effector of K-Ras, in pancreatic cancer that warrants further clinical investigations. Citation Format: Asfar S. Azmi, William Senapedis, Yosef Landesman, Erkan Baloglu, Ori Kalid, Jack Wu, Bin Bao, Amro Aboukameel, Sharon Shacham, Michael Kauffman, Ramzi M. Mohammad. Novel small molecule pak4 allosteric modulators with activity against pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1771. doi:10.1158/1538-7445.AM2014-1771

Abstract Epigenetic alterations, including histone modifications, play fundamental roles in breast cancer initiation and progression. We originally identified and cloned the GASC1 (gene amplified in squamous cell carcinoma 1, also known as KDM4C) gene from an amplified region at 9p24 in esophageal cancer cells; and recently demonstrated that KDM4C/GASC1 is amplified and over-expressed in breast cancer, particularly in the aggressive basal subtype. The KDM4C/GASC1 protein belongs to the KDM4 family of histone demethylases, and although KDM4 family members have a high degree of homology, they may play different roles in various types of breast cancer. The goal of this study is to analyze genomic anomalies and expression levels of KDM4 demethylases in breast cancer, and elucidate the fundamental role and mechanism of their dysregulation in promoting breast tumorigenesis. We conducted a large-scale meta-analysis of KDM4 demethylase expression across multiple available gene expression studies in breast cancer. Next, we examined KDM4 expression in a panel of non-tumorigenic and cancerous breast epithelial cell lines using quantitative RT-PCR and Western blot assays. We also assessed global methylation (H3K4, H3K9, H3K27 and H3K36) levels by Western blot in a panel of breast cancer cell lines. Finally, we tested a novel KDM4 inhibitor in breast cancer. We found that the KDM4 members show different expression patterns in subtypes of breast cancer. GASC1/KDM4C expression is high in estrogen receptor (ER)-negative, basal type breast cancers. In contrast, KDM4B expression is significantly higher in ER-positive luminal-type breast cancers. Expression levels of homologs KDM4A and D are not significantly different between ER-+/- breast cancers. Our findings suggest that H3 global methylation levels vary among different breast cancer cell lines. Furthermore, we demonstrated that inhibition of KDM4 with a novel small molecule inhibitor increased H3K9 methylation levels and slowed KDM4-overexpressed breast cancer cell growth in vitro. In summary, our data indicate that the KDM4 histone demethylase family may contribute to the dysregulation of histone methylation status differently in breast cancer subtypes. Moreover, breast cancer cell lines with defined histone methylation levels will provide a useful model for investigating biological and functional roles of KDM4 histone demethylases, and for developing novel anticancer epi-drugs in breast cancer. Citation Format: Andreana Holowatyj, Qin Ye, Lihong Zhang, Jack Wu, Zeng-Quan Yang. Targeting the histone demethylase KDM4 subfamily as a potential therapeutic strategy in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5154. doi:10.1158/1538-7445.AM2014-5154

Transcription of a pre-mRNA in eukaryotic cells elongates from the 5′ to the 3′ end, but intron removal during a pre-mRNA splicing does not always proceed in this orientation. In this study, we identified eight mouse p53 transcripts that retained one or more of introns 6, 7 and 8. The 5′ part of intron 9 was also retained while the 3′ part was not studied. These intron-containing transcripts, abbreviated as p53-ICTs, were detected at low abundance in many mouse embryonic fibroblasts (MEF) as well as cancer cell lines and tissues, and the highest ratio of these p53-ICTs to the mature p53 mRNA was seen in the normal pancreas. Serum starvation decreased those p53-ICTs that retained introns 6 and 7 but increased the levels of those lacking these introns while the level of the mature p53 mRNA was unaffected. Treatment of several cancer cell lines with cisplatin increased the mature p53 mRNA level but decreased these p53-ICTs. Transfection of p53−/− MEF with the p53 cDNA or several p53-ICT mini-genes slightly increased the cell viability and rendered the cells resistant to cisplatin. These data also suggest that p53 pre-mRNA splicing may have multiple orders of intron removal, some of which may not follow the “first come, first served” principle. It remains possible that these p53-ICTs are splicing intermediates existing as a mechanism for the cell to respond more promptly to a demand for more p53 and that p53 protein may be required for a normal life of MEF.

Lysine-specific demethylase 5A (KDM5A), an enzyme that removes activating H3K4 di- and trimethylation marks, plays critical roles in controlling transcription and chromatin architecture, yet its biological functions largely remain uncharacterized, particularly in the context of human cancer. In the present study, we found that the KDM5A gene was significantly amplified and over-expressed in various human tumors, including breast cancer. Reducing the expression of KDM5A by shRNA knockdown inhibited proliferation of KDM5A-amplified breast cancer cells. More importantly, we demonstrated that KDM5A over-expression was associated with breast cancer drug resistance. Furthermore, knockdown of KDM5A gene expression altered H3K4 methylation and induced upregulation of CDK inhibitors as well as genes mediating apoptotic cell death. Taken together, our study strongly links KDM5A histone demethylase activity to breast cancer proliferation and drug resistance, and suggests KDM5A is a potential target for breast cancer therapy.

Abstract Gene amplification is a major mechanism used by cancer cells to increase gene expression and subsequently gain a growth and survival advantage. Previously, we identified a novel amplicon at 9p23-24 region in human esophageal and breast cancer and originally cloned a novel gene GASC1 (Gene Amplified in Squamous Cell Carcinoma 1) from this amplicon. Accumulating evidence suggests common amplicons that occur in breast and other cancers contain multiple oncogenes that could play a role in cancer initiation and progression. In the current study, we aimed to comprehensively characterize the 9p23-24 amplified genes in human breast cancer. We performed array CGH analysis on a panel of cancer cell lines with the Agilent 244K microarray chip and narrowed the shortest region of overlapping (SRO) of the 9p23-24 amplicon to ∼2 Mb. Eleven genes within the SRO regions were examined for their mRNA expression by quantitative RT-PCR (qRT-PCR). Based on statistical association between copy number and expression, we confirmed GASC1 as a top candidate oncogene and identified three new potential oncogenes, UHRF2, KIAA1432 and C9orf123. Our more recent studies indicated that GASC1 is a member of a new class of oncogenes that are involved in the deregulation of histone methylation in cancer cells. We found that GASC1 induces the expression of core stemness transcription factors NANOG, SOX2 and OCT4, as well as classical oncogenes, including NOTCH1 and MYC, in breast cancer cells. On the other hand, UHRF2 encodes a nuclear protein that is involved in cell-cycle regulation. Knocking down UHRF2 slowed cell growth and inhibited colony formation of 9p23-24 amplified breast cancer cells. Furthermore, we demonstrated that expression levels of key tumor suppressing genes, including p16/INK4a, p21/WAF1 and p27/KIP1, were increased after knocking down UHRF2 in breast cancer cells. Collectively, our studies support the notion that the 9p23-24 amplicon contains multiple candidate oncogenes and may play an important role in human tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4700. doi:10.1158/1538-7445.AM2011-4700

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.

The roles of Se deficiency and Fe atoms, for the superconductivity of FeSe, have been, respectively, investigated in FeSe1− and Fe1−xCux Se1− , by our NMR experiments. The data, for nuclear spin-lattice relaxation rate 1 /T1 , show that the spin fluctuations are weakened at a larger , and are correlated with the superconductivity in FeSe. The superconducting volume fraction, estimated by our ac susceptibility experiments, is found to vary inversely with . Our findings suggest that the Se-deficient FeSe has an inhomogeneous phase, where the superconductivity is associated with the regions having few or no Se vacancies. As for Fe1−xCux Se1− , Tc is rapidly suppressed by Cu doping and vanishes around x=0.03. The Se and the Cu NMR linewidths suggest that a local moment is induced at the Fe sites and not at the Cu sites. However, 1 /T1 shows no obvious change with the Cu doping. We suspect that other effects, such as disorder or change in the density of states, have more influence on Tc suppression since a metal-insulator transition, induced by Cu substitution, occurs in the resistivity measurements.

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.

The roles of Se deficiency and Fe atoms, for the superconductivity of FeSe, have been, respectively, investigated in FeSe1-delta and (Fe1-xCux)Se1-delta, by our NMR experiments. The data, for nuclear spin-lattice relaxation rate (1/T-1), show that the spin fluctuations are weakened at a larger delta, and are correlated with the superconductivity in FeSe. The superconducting volume fraction, estimated by our ac susceptibility experiments, is found to vary inversely with delta. Our findings suggest that the Se-deficient FeSe has an inhomogeneous phase, where the superconductivity is associated with the regions having few or no Se vacancies. As for (Fe1-xCux)Se1-delta, T-c is rapidly suppressed by Cu doping and vanishes around x=0.03. The Se-77 and the Cu-63 NMR linewidths suggest that a local moment is induced at the Fe sites and not at the Cu sites. However, 1/T-1 shows no obvious change with the Cu doping. We suspect that other effects, such as disorder or change in the density of states, have more influence on T-c suppression since a metal-insulator transition, induced by Cu substitution, occurs in the resistivity measurements.

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA.

BACKGROUND:MI-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. RESULTS:MI-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 muM, 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. CONCLUSION:Overall, 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.

c-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.

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-DLCL(2) and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC(50) 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-DLCL(2) 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-X(L) proteins with high affinity and is shown to block the formation of heterodimers between Bcl-X(L) 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 DLCL(2) cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL(2) 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.