Aruna Basu

Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis

Bcl‐xL, a close homolog of Bcl2, is an important regulator of apoptosis and is overexpressed in human cancer. Phosphorylation of Bcl‐xL can be induced by microtubule‐damaging drugs such as taxol or 2‐methoxyestradiol (2‐ME). By site‐directed mutagenesis studies, we have identified that serine 62 is the necessary site for taxol‐ or 2‐ME‐induced Bcl‐xL phosphorylation in prostate cancer cells. Further studies with the inhibitor of Jun kinase (JNK) and phosphorylation null mutant of Bcl‐xL reveal the augmentative role of JNK‐mediated Bcl‐xL phosphorylation in apoptosis of prostate cancer cells. In summary, our studies suggest that the phosphorylation of Bcl‐xL by stress response kinase signaling might oppose the anti‐apoptotic function of Bcl‐xL to permit prostate cancer cells to die by apoptosis.

Crosstalk between Extrinsic and Intrinsic Cell Death Pathways in Pancreatic Cancer: Synergistic Action of Estrogen Metabolite and Ligands of Death Receptor Family

2-Methoxyestradiol is a physiologic metabolite of 17beta-estradiol. This orally active compound can inhibit tumor growth or metastasis in tumor models without inducing any clinical sign of toxicity. Our previous studies indicated that 2-methoxyestradiol-mediated apoptosis involves the disappearance of intact 21-kDa Bid protein, cytochrome c release, and predominant procaspase-3 cleavage. Here, using MIA PaCa-2 cells as a model, we investigated whether this estrogen metabolite induces apoptosis by converging two major pathways: the death receptor-mediated extrinsic and the mitochondrial intrinsic pathway. Exogenous expression of dominant-negative caspase-8 or dominant-negative FADD reverts the effect of 2-methoxyestradiol-mediated cell death. In parallel with this observation, Z-IETD-FMK, a cell permeable irreversible inhibitor of caspase-8, can render significant protection against 2-methoxyestradiol-induced apoptosis. RNase protection assay and cell surface receptor analysis by flow cytometry show the up-regulation of members of death receptor family in 2-methoxyestradiol-exposed pancreatic cancer cells. Our mechanistic studies also implicate that oxidative stress precedes 2-methoxyestradiol-mediated c-Jun NH2-terminal kinase activation, leading to elevated Fas level. Because 2-methoxyestradiol is able to trigger death receptor signaling, we were interested in examining the effects of 2-methoxyestradiol and Fas ligand (FasL)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) together on pancreatic cancer cell death. Interestingly, the endogenous angiogenesis inhibitor 2-methoxyestradiol augments FasL/TRAIL-induced apoptosis in these cells. Moreover, the combination of 2-methoxyestradiol and TRAIL reduces the tumor burden in vivo in MIA PaCa-2 tumor xenograft model by caspase-3 activation.

MicroRNA-375 and MicroRNA-221 Potential Noncoding RNAs Associated with Antiproliferative Activity of Benzyl Isothiocyanate in Pancreatic Cancer

The deregulated presence or absence of microRNAs (miRNAs) might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. In this study, we provide evidence that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Interestingly, ectopic expression of miR-375 or the enforced silencing of miR-221 in cultured pancreatic cancer cells attenuates cell viability and sensitizes antiproliferative action of BITC. We also show that the expression of putative tumor suppressor miR-375 is more abundant in nonpathological mice pancreata than those with Kras(G12D)-driven pancreatic intraepithelial neoplasia (PanIN). To the contrary, the expression of oncogenic miR-221 is significantly elevated in the mouse pancreas with PanIN lesions. Although miR-375 has been shown to be aberrantly expressed in pancreatic cancer patients, there has not been a comprehensive study to investigate the molecular pathways targeted by this miRNA in pancreatic cancer cells. Further analysis by gene expression microarray revealed that IGFBP5 and CAV-1, potential biomarkers of pancreatic cancer, were significantly downregulated in cells transfected with miR-375. Correlatively, elevated expression of IGFBP5 and CAV-1 was evident in the mouse pancreas with preneoplastic lesions in which the expression of miR-375 wanes. Taken together, our findings suggest that anticancer agent BITC might target the expression of miR-221 and miR-375 to switch hyperproliferative pancreatic cancer cells to a hypoproliferative state.

2-Methoxyestradiol mediated signaling network in pancreatic cancer.

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17 beta-estradiol, is known to be a potent inhibitor of neovascularization. Our previous studies have shown that 2-ME can suppress growth of pancreatic tumor cells in vitro and in vivo by the induction of apoptosis (Cancer Res 66: 4309-18, 2006). In order to better comprehend the signaling modulators of 2-ME in pancreatic cancer, we employed a PowerBlot Western Array screening system. Our proteomic profiling has provided framework to define the novel mechanisms of actions of 2-ME in pancreatic cancer. Interestingly, this high-throughput analysis identified proteins such as Rac1, Gelsolin, Glucocorticoid receptor (GR), Smad 2/3, Smad 4, IRS-1, which were not previously reported with 2-ME response. Interestingly, 2-ME modulated down regulation of GR level is accompanied by NF-k B activation in 2-ME responsive but not in resistant pancreatic cancer cells. In view of this observation, possible reciprocal relationship between GR and NF-kappaB activation might be an important regulatory factor in 2-ME mediated demise of a subpopulation of pancreatic cancer cells.

Abstract 15: Phosphorylation defective mutant Bcl-xL knockin mice exhibit behavioral abnormalities linked with neurodevelopmental disorders

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Bcl-xL, a close homolog of Bcl2, is an important regulator of apoptosis and is overexpressed in many human cancers including neuroblastoma. Previously we have discovered that the regulation of phosphorylation of Bcl-xL on serine 62 residue by tubulin-binding anticancer drugs at mitosis may function as a checkpoint to permit apoptosis of cancer cells. However, the physiological significance of Bcl-xL phosphorylation still remains obscure. With a view to comprehending the functional relevance of Bcl-xL phosphorylation in vivo, we have generated knock-in mice expressing an unphosphorylable ser62 (equivalent to human ser62), by substitution with an alanine residue. Thus placing the phosphorylation-defective mutant of Bcl-xL into the germ line of mice creates opportunities to observe the effects of endogenous phosphorylation in the context of multicellular organisms. Since Bcl-xL has been shown to be maintained at high level in CNS, initially a full behavioral phenotyping battery was carried out with wild type (WT) and Bcl-xLS62A(+/+) mice. Born at Mendelian ratios, the Bcl-xLS62A(+/+) mice demonstrated reduced startle response and reduced PPI (Prepulse inhibition) at lower PPI amplitudes compared to WT mice. Moreover, cognitive deficits were noted in the passive avoidance task between Bcl-xLS62A(+/+) mice and WT littermates. These data indicate that mutation in the phosphorylation site of Bcl-xL drive a range of functional abnormalities and many of the observed behavioral abnormalities in Bcl-xLS62A(+/+) mice are consistent with neurodevelopmental disorders. In all, our findings suggest that endogenous phosphorylation of antiapoptotic protein Bcl-xL might play a critical role in neurodevelopment and subsequent CNS functional output. Further molecular analysis on the effect of blocking of endogenous phosphorylation of Bcl-xL on neuronal apoptosis will be discussed. 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 15. doi:10.1158/1538-7445.AM2011-15

Abstract 139: MicroRNA-375 and microRNA-221: Potential noncoding RNAs associated with antiproliferative activity of benzyl isothiocyanate in pancreatic cancer

The deregulated presence or absence of microRNAs (miRNAs) might play an important role in molecular pathways leading to neoplastic transformation. At present, it is also thought that the approaches to interfere miRNA functions should be helpful for developing novel therapeutic opportunities for human cancer. In this study, we provide evidence that the anticancer agent benzyl isothiocyanate (BITC) has the ability to modulate the level of miRNAs such as miR-221 and miR-375, known to be abnormally expressed in pancreatic cancer patients. Interestingly, ectopic expression of miR-375 or the enforced silencing of miR-221 in cultured pancreatic cancer cells attenuates cell viability and sensitizes antiproliferative action of BITC. We also show that the expression of putative tumor suppressor miR-375 is more abundant in nonpathological mice pancreata than those with Kras G12D -driven pancreatic intraepithelial neoplasia (PanIN). To the contrary, the expression of oncogenic miR-221 is significantly elevated in the mouse pancreas with PanIN lesions. Although miR-375 has been shown to be aberrantly expressed in pancreatic cancer patients, there has not been a comprehensive study to investigate the molecular pathways targeted by this miRNA in pancreatic cancer cells. Further analysis by gene expression microarray revealed that IGFBP5 and CAV-1, potential biomarkers of pancreatic cancer, were significantly downregulated in cells transfected with miR-375. Correlatively, elevated expression of IGFBP5 and CAV-1 was evident in the mouse pancreas with preneoplastic lesions in which the expression of miR-375 wanes. Taken together, our findings suggest that anticancer agent BITC might target the expression of miR-221 and miR-375 to switch hyperproliferative pancreatic cancer cells to a hypoproliferative state.