Usha Kasid

Identification of a Novel Tumor Necrosis Factor-α-inducible Gene, SCC-S2, Containing the Consensus Sequence of a Death Effector Domain of Fas-associated Death Domain-like Interleukin- 1β-converting Enzyme-inhibitory Protein

We report here the isolation and characterization of a novel tumor necrosis factor-α (TNF-α)-inducible gene,SCC-S2. Based on the nucleotide sequence, the SCC-S2 open reading frame contains a sequence in the amino terminus that shows a significant homology to death effector domain II of cell death regulatory protein, Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein (FLIP). Unlike FLIP, the SCC-S2 open reading frame contains only one death effector domain and lacks the carboxyl-terminal caspase-like homology domain, raising the possibility that SCC-S2 may be a novel member of the FLIP family. SCC-S2 mRNA expression is found in most normal tissues and malignant cells. The steady state level ofSCC-S2 mRNA is significantly induced by TNF-α in different tumor cells (TNF-α at 20 ng/ml for 3 h: A549, ∼2–9-fold; SKOV-3, ∼3-fold; PCI-04A, ∼3–6-fold). TNF-α treatment (100 ng/ml, 4 h) of HeLa cells transiently transfected with FLAG epitope-tagged SCC-S2 cDNA or expression vector alone led to an increase in the number of apoptotic cells as compared with the untreated counterpart. Interestingly, however,SCC-S2 transfectants revealed a significant decrease in the number of apoptotic cells as compared with the vector transfectants (p < 0.001). These data implicate a role of SCC-S2 as a negative mediator of apoptosis in certain cell types.

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice.

In previous studies, nitroxide tempo (2, 2, 6, 6‐tetramethyl‐piperidine‐1‐oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone‐dependent/hormone‐independent prostate carcinoma models (LNCaP, DU‐145, and PC‐3).

The significance of TNFAIP8 in prostate cancer response to radiation and docetaxel and disease recurrence

TNFAIP8 is a NF‐κB‐inducible, oncogenic molecule. Previous “promoter array” studies have identified differential methylation and regulation of TNFAIP8 in prostate epithelial and cancer cell lines. Here we demonstrate that TNFAIP8 expression is induced by androgen in hormone‐responsive LNCaP prostate cancer cells. In athymic mice bearing hormone‐refractory PC‐3 prostate tumor xenografts, intravenous treatment with a liposomal formulation of TNFAIP8 antisense oligonucleotide (LE‐AS5) caused reduced expression of TNFAIP8 in tumor tissues, and a combination of LE‐AS5 and radiation or docetaxel treatment resulted in significant inhibition of PC‐3 tumor growth as compared to single agents. The immunohistochemical evaluation of TNFAIP8 expression revealed correlation of both cytoplasmic and nuclear TNFAIP8 overexpression with high grade prostatic adenocarcinomas, while nuclear overexpression was found to be an independent predictor of disease recurrence controlling for tumor grade. Increased nuclear TNFAIP8 expression was statistically significantly associated with a 2.44 fold (95 % confidence interval: 1.01–5.91) higher risk of prostate cancer recurrence. Mechanistically, TNFAIP8 seems to function as a scaffold (or adaptor) protein. In the antibody microarray analysis of proteins associated with the TNFAIP8 immune‐complex, we have identified Karyopherin alpha2 as a novel binding partner of nuclear TNFAIP8 in PC‐3 cells. The Ingenuity Pathway Analysis of the TNFAIP8 interacting proteins suggested that TNFAIP8 influences cancer progression pathways and networks involving integrins and matrix metalloproteinases. Taken together, present studies demonstrate that TNFAIP8 is a novel therapeutic target in prostate cancer, and indicate a potential relationship of the nuclear trafficking of TNFAIP8 with adverse outcomes in a subset of prostate cancer patients.

Ionizing radiation stimulates octamer factor DNA binding activity in human carcinoma cells.

In mammalian cells, the octamer motif (ATGCAAAT) binding proteins, Oct-1 and Oct-2, play an important role in the transcriptional transactivation of several ubiquitously expressed genes as well as cell-specifically expressed genes. To date, a role of the octamer binding proteins in damage-stimulated response is not known. In this report, we demonstrate that DNA-binding activity of Oct-1, as demonstrated by the electrophoretic mobility shift assay, is significantly induced in a dose-dependent manner upon treatment of human head and neck squamous carcinoma cells (PCI-04A) with ionizing radiation (5 Gy: 5-fold; 15 Gy: 11-fold). By comparison, activities of other transcription factors were modestly increased (15 Gy: AP-1, 2.5-fold; NF-κB, 2.6-fold; SP-1, 5-fold). Radiation stimulation of Oct-1 activity was also noted in two other human cancer cell lines, albeit to a lesser extent (MDA-MB231 breast carcinoma cells and PC-3 prostate carcinoma cells (5 Gy: ∼ 2-fold). These data represent the first report of the activation of an octamer factor DNA binding activity in response to environmental cues and suggest a novel role of Oct-1 in the radiation signaling cascade in these cancer cells.

Antisense oligonucleotides: target validation and development of systemically delivered therapeutic nanoparticles.

Antisense oligonucleotides (ASO) against specific molecular targets (e.g., Bcl-2 and Raf-1) are important reagents in cancer biology and therapy. Phosphorothioate modification of the ASO backbone has resulted in an increased stability of ASO in vivo without compromising, in general, their target selectivity. Although the power of antisense technology remains unsurpassed, dose-limiting side effects of modified ASO and inadequate penetration into the tumor tissue have necessitated further improvements in ASO chemistry and delivery systems. Oligonucleotide delivery systems may increase stability of the unmodified or minimally modified ASO in plasma, enhance uptake of ASO by tumor tissue, and offer an improved therapy response. Here, we provide an overview of ASO design and in vivo delivery systems, and focus on preclinical validation of a liposomal nanoparticle containing minimally modified raf antisense oligodeoxynucleotide (LErafAON). Intact rafAON (15-mer) is present in plasma and in normal and tumor tissues of athymic mice systemically treated with LErafAON. Raf-1 expression is decreased in normal and tumor tissues of LErafAON-treated mice. Therapeutic benefit of a combination of LErafAON and radiation or an anticancer drug exceeds radiation or drug alone against human prostate, breast, and pancreatic tumors grown in athymic mice. Further improvements in ASO chemistry and nanoparticles are promising avenues in antisense therapy of cancer.

Ionizing Radiation and TNF-a and Stimulated Expression of a1-Antichymotrypsin Gene in Human Squamous Carcinoma Cells

Alpha-1 antichymotrypsin (ACT), a serine protease inhibitor, has been detected in several epithelial tumor cell types, but its role in response to therapy is not clear. We report here that exposure of primary head and neck squamous cell carcinoma (HNSCC)-derived cells (PCI-04A) to ionizing radiation (IR) or tumor necrosis factor-alpha (TNF-alpha) resulted in an increased level of ACT mRNA, although the induction patterns were different. IR treatment caused a transient stimulation of ACT mRNA, peaking at 3 h post-irradiation, whereas TNF-alpha-inducible ACT gene expression lasted for up to 24 h. The ACT mRNA was expressed in several epithelial and non-epithelial tumor cell types, and in different normal human tissues. In addition, when the ACT gene expression in PCI-04A cells was compared with the matched (from the same patient) metastatic HNSCC-derived cells (PCI-04B), increased steady-state level of the ACT mRNA was observed in PCI-04B cells. Taken together, these findings suggest that ACT may serve as an important marker for prognosis and therapy selection in HNSCC.

Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Stress-responsive signal transduction: emerging concepts and biological significance

In the past few years, substantial evidence has been put forth that suggests increasing complexity of the mechanism of action of cytotoxic, mutagenic or carcinogenic agents, commonly known as stress-inducers. The developing theme in a stress-induced cellular response is stimulation of a signal transduction pathway regulated by either cell growth and proliferation-related (survival) factors or programmed cell death-related (apoptotic) factors. The balance between the survival and apoptotic signals depends on the cell type, and dictates the fate of the cell. This review summarises current knowledge of the intracellular signal transduction responses elicited by ionising radiation in rodent and mammalian cells, and focuses on the involvement of Raf-1 protein serine-threonine kinase in the signalling pathways initiated by diverse stress-inducers including ionising radiation, ultraviolet radiation, growth factor-deprivation, and cytokines.

TMEM33: a new stress-inducible endoplasmic reticulum transmembrane protein and modulator of the unfolded protein response signaling

Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) signaling cascade and induction of an apoptotic cell death, autophagy, oncogenesis, metastasis, and/or resistance to cancer therapies. Mechanisms underlying regulation of ER transmembrane proteins PERK, IRE1α, and ATF6α/β, and how the balance of these activities determines outcome of the activated UPR, remain largely unclear. Here, we report a novel molecule transmembrane protein 33 (TMEM33) and its actions in UPR signaling. Immunoblotting and northern blot hybridization assays were used to determine the effects of ER stress on TMEM33 expression levels in various cell lines. Transient transfections, immunofluorescence, subcellular fractionation, immunoprecipitation, and immunoblotting were used to study the subcellular localization of TMEM33, the binding partners of TMEM33, and the expression of downstream effectors of PERK and IRE1α. Our data demonstrate that TMEM33 is a unique ER stress-inducible and ER transmembrane molecule, and a new binding partner of PERK. Exogenous expression of TMEM33 led to increased expression of p-eIF2α and p-IRE1α and their known downstream effectors, ATF4-CHOP and XBP1-S, respectively, in breast cancer cells. TMEM33 overexpression also correlated with increased expression of apoptotic signals including cleaved caspase-7 and cleaved PARP, and an autophagosome protein LC3II, and reduced expression of the autophagy marker p62. TMEM33 is a novel regulator of the PERK-eIE2α-ATF4 and IRE1-XBP1 axes of the UPR signaling. Therefore, TMEM33 may function as a determinant of the ER stress-responsive events in cancer cells.

The association of human c-Ha-ras sequences with chromatin and nuclear proteins

As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells. This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences. The polynucleosomal fraction was further resolved by sucrose gradient sedimentation. Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction. (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging. Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined. The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.