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/ß, 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 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.

Restricted expression of new guanine nucleotide exchange factor Zizimin2 in aged acquired immune system.

The activity of various biological functions, such as nervous, endocrine and immune systems including acquired immunity, is known to decline along with aging. To elucidate the molecular mechanism of this phenomenon, we here compared the number of thymocytes, splenocytes, and bone marrow lymphocytes in young and aged mice and found the age-related functional fragility of the immune system. However, the molecular mechanisms or even the key molecules remain elusive. Therefore, we further focused on a candidate for immunosenesence-related molecules, Zizimin2, which we have recently isolated and identified as a novel guanine nucleotide exchange factor that is highly expressed in murine splenic germinal center B cells after immunization with a T cell-dependent antigen. Here, we showed that endogenous Zizimin2 protein as well as mRNA expression levels in immune organs are strictly suppressed in aged mice. We further observed that the serum antigen specific antibody response is hampered in aged mice compared to that in young animals. Moreover, the Zizimin2 mRNA expression level was not activated after immunization in aged mice. Taken together, these data suggested that Zizimin2 is associated with the reduction of immune response in acquired immunity along with aging.

Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells.

BACKGROUND: We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cells after immunization with T-cell-dependent antigen. Zizimin2 was revealed to be a new family member of Dock (dedicator of cytokinesis), Dock11, which is the guanine nucleotide exchange factor for Cdc42, a low-molecular-weight GTPase. However, the molecular function of Zizimin2 in acquired immunity has not been elucidated. RESULTS: In this study, we show that the protein expression of Zizimin2, which is also restricted to lymphoid tissues and lymphocytes, is reduced in aged mice. Over-expression of full-length Zizimin2 induced filopodial formation in 293T cells, whereas expression of CZH2 domain inhibited it. Stimulation of Fcγ receptor and Toll-like receptor 4 triggered Zizimin2 up-regulation and Cdc42 activation in bone marrow-derived dendritic cells. CONCLUSIONS: These data suggest that Zizimin2 is an immune-related and age-regulated guanine nucleotide exchange factor, which facilitates filopodial formation through activation of Cdc42, which results in activation of cell migration.

Rat monoclonal antibodies against new guanine nucleotide exchange factor, mouse Zizimin2.

We recently isolated and identified Zizimin2 as a functional factor that is highly expressed in murine splenic germinal center B cell after immunization with T-cell-dependent antigen. Zizimin2 was revealed that it is a new family member of Dock (dedicator of cytokinesis), Dock11, which was the guanine nucleotide exchange factor for Cdc42, the Rho family of low molecular weight GTPases. However, neither the molecular function of Zizimin2 in the acquired immunity nor the physiological role in decrease of immunological activity along with aging has been known. To facilitate the further analysis of physiological function of this molecule, we have established rat monoclonal antibodies (MAbs) against mouse Zizimin2 protein. Recombinant His-tagged partial mouse Zizimin2 protein was expressed in Escherichia coli, purified, and applied as an antigen for immunization. The desired hybridomas were selected by the recombinant Zizimin2 protein coated enzyme-linked immunosorbent assay screening, and we generated stable hybridoma cell lines that produced the antibody against murine Zizimin2 protein. We determined their isotypes and further examined capability or availability in immunoblotting, immunoprecipitation, or immunofluorescence microscopy. Here we demonstrated the several appropriate antibodies for immunoblotting and immunofluorescence microscopy. These MAbs should therefore be very useful tools for study of the characterization of the Zizimin2 protein and the understanding of the biological function of Zizimin2-mediated immunosenescence.

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.

Role of SCC-S2 in experimental metastasis and modulation of VEGFR-2, MMP-1, and MMP-9 expression.

SCC-S2/GG2-1/NDED (approved gene symbol TNFAIP8) is a transcription factor NF-kappaB-inducible, antiapoptotic, and oncogenic molecule. In this study, we examined the role of SCC-S2 in invasion and experimental metastasis. We demonstrate that expression of SCC-S2 cDNA in MDA-MB 435 human breast cancer cells is associated with enhanced invasion in vitro and increased frequency of pulmonary colonization of tumor cells in athymic mice. Systemic treatment of athymic mice with a cationic liposomal formulation of SCC-S2 antisense oligo led to decreased incidence of pulmonary metastasis and inhibition of SCC-S2 expression in vivo. Antisense inhibition of endogenous SCC-S2 expression correlated with decreased expression of VEGF receptor-2 in tumor cells and human lung microvascular endothelial cells and loss of endothelial cell viability. In addition, downregulation of SCC-S2 expression in tumor cells was associated with decreased expression of known metastasis-related molecules MMP-1 and MMP-9. These results demonstrate a novel role for SCC-S2 in tumor progression, involving multiple effectors, and provide a basis for SCC-S2-targeted cancer gene therapy.

Systemic delivery of RafsiRNA using cationic cardiolipin liposomes silences Raf-1 expression and inhibits tumor growth in xenograft model of human prostate cancer.

Raf-1, a protein serine-threonine kinase, plays a critical role in mitogen-activated protein kinase kinase (MKK/MEK)- mitogen-activated protein kinase (extracellular signal-regulated kinase) (MAPK/ERK) pathways. We show here that systemically delivered novel cationic cardiolipin liposomes (NeoPhectin-AT) containing a small interfering RNA (siRNA) against Raf-1 silence the expression of Raf-1 in tumor tissues and inhibit tumor growth in xenograft model of human prostate cancer. The knockdown of Raf-1 expression by siRNA is also associated with down-regulation of cyclin D1 expression in vivo.

SCC-112, a novel cell cycle-regulated molecule, exhibits reduced expression in human renal carcinomas.

We report here the identification and an initial characterization of a novel cell cycle-regulated molecule, SCC-112. SCC-112 cDNA (6744 bp) encodes a longest open reading frame (ORF) comprised of 1297 amino acids, representing a approximately 150-kDa nuclear protein. SCC-112 mRNA and protein levels were relatively high during the G2/M phase of the cell cycle in MDA-MB 435 breast cancer cells. Transient expression of SCC-112 cDNA in COS-1 cells led to an increase in the number of cells in sub-G1 phase and enhanced activity of caspase-3, a downstream effector of apoptosis. Stable transfection of SCC-112 cDNA in MDA-MB 231 breast cancer cells also led to an increase in the number of cells in sub-G1 phase ( approximately 2-3-fold), indicative of apoptosis. The examination of the paired sets of human normal and tumor tissues revealed that the SCC-112 mRNA level was significantly high in normal breast and kidney tissues as compared to the corresponding primary tumor tissues (P<0.0001; breast, n=50, and kidney, n=20). Consistent with these observations, SCC-112 protein expression (150 kDa) was high in a majority of the normal renal tissues examined as compared to the matched renal tumor tissues (67%, 1.2-fold to>10-fold, n=18). Taken together, these findings suggest that the SCC-112 gene expression is likely to be associated with normal cell growth and proliferation.