CCAR-1 works together with the U2AF large subunit UAF-1 to regulate alternative splicing.

The Cell Division Cycle and Apoptosis Regulator (CCAR) protein family members have recently emerged as regulators of alternative splicing and transcription, as well as having other key physiological functions. For example, mammalian CCAR2/DBC1 forms a complex with the zinc factor protein ZNF326 to integrate alternative splicing with RNA polymerase II transcriptional elongation in AT-rich regions of the DNA. Additionally, Caenorhabditis elegans CCAR-1, a homolog to mammalian CCAR2, facilitates the alternative splicing of the perlecan unc-52 gene. However, much about the CCAR family's role in alternative splicing is unknown. Here, we have examined the role of CCAR-1 in genome-wide alternative splicing in Caenorhabditis elegans and have identified new alternative splicing targets of CCAR-1 using RNA sequencing. Also, we found that CCAR-1 interacts with the spliceosome factors UAF-1 and UAF-2 using mass spectrometry, and that knockdown of ccar-1 affects alternative splicing patterns, motility, and proteostasis of UAF-1 mutant worms. Collectively, we demonstrate the role of CCAR-1 in regulating global alternative splicing in C. elegans and in conjunction with UAF-1.

Deregulated expression of the 14q32 miRNA cluster in clear cell renal cancer cells.

Clear cell renal cell carcinomas (ccRCC) are characterized by arm-wide chromosomal alterations. Loss at 14q is associated with disease aggressiveness in ccRCC, which responds poorly to chemotherapeutics. The 14q locus contains one of the largest miRNA clusters in the human genome; however, little is known about the contribution of these miRNAs to ccRCC pathogenesis. In this regard, we investigated the expression pattern of selected miRNAs at the 14q32 locus in TCGA kidney tumors and in ccRCC cell lines. We demonstrated that the miRNA cluster is downregulated in ccRCC (and cell lines) as well as in papillary kidney tumors relative to normal kidney tissues (and primary renal proximal tubule epithelial (RPTEC) cells). We demonstrated that agents modulating expression of DNMT1 (e.g., 5-Aza-deoxycytidine) could modulate 14q32 miRNA expression in ccRCC cell lines. Lysophosphatidic acid (LPA, a lysophospholipid mediator elevated in ccRCC) not only increased labile iron content but also modulated expression of a 14q32 miRNA. Through an overexpression approach targeting a subset of 14q32 miRNAs (specifically at subcluster A: miR-431-5p, miR-432-5p, miR-127-3p, and miR-433-3p) in 769-P cells, we uncovered changes in cellular viability and claudin-1, a tight junction marker. A global proteomic approach was implemented using these miRNA overexpressing cell lines which uncovered ATXN2 as a highly downregulated target. Collectively, these findings support a contribution of miRNAs at 14q32 in ccRCC pathogenesis.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

Lipid mediators and biomarkers associated with type 1 diabetes development.

Type 1 diabetes (T1D) is a consequence of autoimmune ß cell destruction, but the role of lipids in this process is unknown. We previously reported that activation of Ca2+-independent phospholipase A2ß (iPLA2ß) modulates polarization of macrophages (MΦ). Hydrolysis of the sn-2 substituent of glycerophospholipids by iPLA2ß can lead to the generation of oxidized lipids (eicosanoids), pro- and antiinflammatory, which can initiate and amplify immune responses triggering ß cell death. As MΦ are early triggers of immune responses in islets, we examined the impact of iPLA2ß-derived lipids (iDLs) in spontaneous-T1D prone nonobese diabetic mice (NOD), in the context of MΦ production and plasma abundances of eicosanoids and sphingolipids. We find that (a) MΦNOD exhibit a proinflammatory lipid landscape during the prediabetic phase; (b) early inhibition or genetic reduction of iPLA2ß reduces production of select proinflammatory lipids, promotes antiinflammatory MΦ phenotype, and reduces T1D incidence; (c) such lipid changes are reflected in NOD plasma during the prediabetic phase and at T1D onset; and (d) importantly, similar lipid signatures are evidenced in plasma of human subjects at high risk for developing T1D. These findings suggest that iDLs contribute to T1D onset and identify select lipids that could be targeted for therapeutics and, in conjunction with autoantibodies, serve as early biomarkers of pre-T1D.

Macrophage polarization is linked to Ca2+-independent phospholipase A2ß-derived lipids and cross-cell signaling in mice.

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)ß (iPLA2ß). Here, we assessed the link between iPLA2ß-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2ß-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2ß.Tg mice with selective iPLA2ß overexpression in ß-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2ß-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2ß.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2ß-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that ß-cell iPLA2ß-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

Serine/Arginine-Rich Splicing Factor 3 Modulates the Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2.

Triple negative breast cancer (TNBC) has an unusually low 5-year survival rate linked to higher metastatic rates. Our laboratory recently delineated a role for the alternative RNA splicing (AS) of cytoplasmic polyadenylation element binding protein 2 (CPEB2), via inclusion/exclusion of exon 4, in the metastasis of TNBC. In these studies, the mechanism governing the inclusion/exclusion of exon 4 was examined. Specifically, the RNA trans-factor, SRSF3, was found to be explicitly associated with CPEB2 exon 4. A SRSF3 consensus sequence was identified in exon 4, and mutation of this sequence abolished the association of SRSF3. The expression of SRSF3 was upregulated in TNBC cells upon the acquisition of anoikis resistance correlating with a reduction in the CPEB2A/B ratio. Importantly, downregulation of SRSF3 in these cells by siRNA induced the exclusion of exon 4 in cells increasing the ratio of CPEB2A (exon 4 excluded) to CPEB2B (exon 4 included). Downregulation of SRSF3 also reversed the CPEB2A/B ratio of a wild-type CPEB2 exon 4 minigene and endogenous CPEB2 pre-mRNA, but not a mutant CPEB2 minigene with the SRSF3 RNA cis-element ablated. SRSF3 downregulation ablated the anoikis resistance of TNBC cells, which was "rescued" by ectopic expression of CPEB2B. Finally, analysis of The Cancer Genome Atlas database showed a positive relationship between SRSF3 expression and lower CPEB2A/B ratios in aggressive breast cancers. IMPLICATIONS: These findings demonstrate that SRSF3 modulates CPEB2 AS to induce the expression of the CPEB2B isoform that drives TNBC phenotypes correlating with aggressive human breast cancer. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/9/1920/F1.large.jpg.

Splice variants of cytosolic polyadenylation element-binding protein 2 (CPEB2) differentially regulate pathways linked to cancer metastasis.

The translational regulator cytosolic polyadenylation element-binding protein 2 (CPEB2) has two isoforms, CPEB2A and CPEB2B, derived by alternative splicing of RNA into a mature form that either includes or excludes exon 4. Previously, we reported that this splicing event is highly dysregulated in aggressive forms of breast cancers, which overexpress CPEB2B. The loss of CPEB2A with a concomitant increase in CPEB2B was also required for breast cancer cells to resist cell death because of detachment (anoikis resistance) and metastasize in vivo To examine the mechanism by which CPEB2 isoforms mediate opposing effects on cancer-related phenotypes, we used next generation sequencing of triple negative breast cancer cells in which the isoforms were specifically down-regulated. Down-regulation of the CPEB2B isoform inhibited pathways driving the epithelial-to-mesenchymal transition and hypoxic response, whereas down-regulation of the CPEB2A isoform did not have this effect. Examining key nodes of these pathways showed that CPEB2B induced the expression of regulatory DNA trans-factors (e.g. HIF1α and TWIST1). Specifically, CPEB2B functioned as a translational activator of TWIST1 and HIF1α. Functional studies showed that specific down-regulation of either HIF1α or TWIST1 inhibited the ability of CPEB2B to induce the acquisition of anoikis resistance and drive metastasis. Overall, this study demonstrates that CPEB2 alternative splicing is a major regulator of key cellular pathways linked to anoikis resistance and metastasis.

PRMT2 interacts with splicing factors and regulates the alternative splicing of BCL-X.

Protein arginine N-methyltransferase 2 (PRMT2) functions in JAK-STAT and Wnt/ß-catenin signalling pathways, serves as a nuclear receptor-dependent transcriptional co-activator, and represses NF-κB and E2F1 transcription factor activities to promote apoptosis. We have previously demonstrated that PRMT2 interacts with PRMT1 and increases its activity. Here, we reveal associations using proteomics between the PRMT2 SH3 domain and splicing factors including Src-associated in mitosis 68 kDa protein (SAM68), a PRMT1 substrate and trans-acting factor that mediates BCL-X alternative splicing. We determined that PRMT2 interacts with SAM68 in cells and regulates its subcellular localization via the SH3 domain of PRMT2, prompting us to investigate the potential role of PRMT2 in BCL-X alternative splicing. We found that the expression of the full-length, wildtype form of PRMT2 promotes an increase in the BCL-X(L)/BCL-X(s) ratio in TNF-α or LPS stimulated cells. These results indicate that active PRMT2 may play a role during inflammation in alternative splicing regulation.

Melanoma Differentiation-associated Gene 7/IL-24 Exerts Cytotoxic Effects by Altering the Alternative Splicing of Bcl-x Pre-mRNA via the SRC/PKCδ Signaling Axis.

Melanoma differentiation-associated gene 7 (MDA-7/IL-24) exhibits cytotoxic effects on tumor cells while sparing untransformed cells, and Bcl-x(L) is reported to efficiently block the induction of cell death by MDA-7/IL-24. The expression of Bcl-x(L) is regulated at the level of RNA splicing via alternative 5' splice site selection within exon 2 to produce either the pro-apoptotic Bcl-x(s) or the anti-apoptotic Bcl-x(L). Our laboratory previously reported that Bcl-x RNA splicing is dysregulated in a large percentage of human non-small cell lung cancer (NSCLC) tumors. Therefore, we investigated whether the alternative RNA splicing of Bcl-x pre-mRNA was modulated by MDA-7/IL-24, which would suggest that specific NSCLC tumors are valid targets for this cytokine therapy. Adenovirus-delivered MDA-7/IL-24 (Ad.mda-7) reduced the viability of NSCLC cells of varying oncogenotypes, which was preceded by a decrease in the ratio of Bcl-x(L)/Bcl-x(s) mRNA and Bcl-x(L) protein expression. Importantly, both the expression of Bcl-x(L) and the loss of cell viability were "rescued" in Ad.mda-7-treated cells incubated with Bcl-x(s) siRNA. In addition, NSCLC cells ectopically expressing Bcl-x(s) exhibited significantly reduced Bcl-x(L) expression, which was again restored by Bcl-x(s) siRNA, suggesting the existence of a novel mechanism by which Bcl-x(s) mRNA restrains the expression of Bcl-x(L). In additional mechanistic studies, inhibition of SRC and PKCδ completely ablated the ability of MDA-7/IL-24 to reduce the Bcl-x(L)/(s) mRNA ratio and cell viability. These findings show that Bcl-x(s) expression is an important mediator of MDA-7/IL-24-induced cytotoxicity requiring the SRC/PKCδ signaling axis in NSCLC cells.

Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis.

Alternate RNA processing of caspase-9 generates the splice variants caspase 9a (C9a) and caspase 9b (C9b). C9b lacks a domain present in C9a, revealing a tumorigenic function that drives the phenotype of non-small cell lung cancer (NSCLC) cells. In this study, we elucidated the mechanistic underpinnings of the malignant character of this splice isoform. In NSCLC cells, C9b expression correlated with activation of the canonical arm of the NF-κB pathway, a major pathway linked to the NSCLC tumorigenesis. Mechanistic investigations revealed that C9b activates this pathway via direct interaction with cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b, but did not require proteolytic cleavage of C9b. This protein:protein interaction was essential for C9b to promote viability and malignant growth of NSCLC cells in vitro and in vivo, broadly translating to diverse NSCLC oncogenotypes. Overall, our findings identified a novel point for therapeutic invention in NSCLC that may be tractable to small-molecule inhibitors, as a new point to broadly address this widespread deadly disease. Cancer Res; 76(10); 2977-89. ©2016 AACR.

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) represents an anomalous subset of breast cancer with a greatly reduced (30%) 5-year survival rate. The enhanced mortality and morbidity of TNBC arises from the high metastatic rate, which requires the acquisition of AnR, a process whereby anchorage-dependent cells become resistant to cell death induced by detachment. In this study TNBC cell lines were selected for AnR, and these cell lines demonstrated dramatic enhancement in the formation of lung metastases as compared with parental cells. Genetic analysis of the AnR subclones versus parental cells via next generation sequencing and analysis of global alternative RNA splicing identified that the mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was altered in AnR TNBC cells. Specifically, increased inclusion of exon 4 into the mature mRNA to produce the CPEB2B isoform was observed in AnR cell lines. Molecular manipulations of CPEB2 splice variants demonstrated a key role for this RNA splicing event in the resistance of cells to anoikis. Specifically, down-regulation of the CPEB2B isoform using siRNA re-sensitized the AnR cell lines to detachment-induced cell death. The ectopic expression of CPEB2B in parental TNBC cell lines induced AnR and dramatically increased metastatic potential. Importantly, alterations in the alternative splicing of CPEB2 were also observed in human TNBC and additional subtypes of human breast cancer tumors linked to a high metastatic rate. Our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key mechanism in AnR and a driving force in TNBC metastasis.

Cystine dimethyl ester induces apoptosis through regulation of PKC-δ and PKC-ε in prostate cancer cells.

Protein kinase C-δ (PKC-δ) and PKC-ε are reported to be effective in cancer prevention via S-thiolation-mediated mechanisms. This may be through stimulation of the pro-apoptotic, tumor-suppressive isozyme PKC-δ and/or inactivation of the growth stimulatory, oncogenic isozyme PKC-ε. We investigated oxidative regulatory responses of PKC-δ and PKC-ε to cystine dimethyl ester (CDME), a metabolic precursor of cystine, which, by inducing release of cellular cystine stimulates apoptosis in different prostate cancer cells, PC3 and LNCaP, compared to normal RWPE1 cells. Treatment of CDME in doses of 0.5mM and 5mM significantly induces apoptosis due to regulation of concentration-dependent PKC-δ stimulation and PKC-ε reduction in these prostate cancer cells. This apoptotic regulation was confirmed by immunoblot analyses and specific PKC enzyme assays in immunoprecipitated samples. Additionally, inhibition of PKC-δ by small interfering RNA (siRNA) proved that CDME-induced cell death was dependent on PKC-δ activity in prostate cancer cells. These data demonstrated that CDME induces apoptosis by cysteinylation of both PKC-δ and PKC-ε in tumorigenic prostate epithelial cells compared to control nontumorigenic cells. Cellular cystine may play a critical role in treatment and/or prevention of prostate cancer by regulating PKC activity.

Combining histone deacetylase inhibitors with MDA-7/IL-24 enhances killing of renal carcinoma cells.

In the present study we show that histone deacetylase inhibitors (HDACIs) enhance the anti-tumor effects of melanoma differentiation associated gene-7/interleukin 24 (mda- 7/IL-24) in human renal carcinoma cells. Similar data were obtained in other GU tumor cells. Combination of these two agents resulted in increased autophagy that was dependent on expression of ceramide synthase 6, with HDACIs enhancing MDA-7/IL-24 toxicity by increasing generation of ROS and Ca (2+). Knock down of CD95 protected cells from HDACI and MDA-7/IL-24 lethality. Sorafenib treatment further enhanced (HDACI + MDA-7/IL-24) lethality. Anoikis resistant renal carcinoma cells were more sensitive to MDA-7/IL-24 that correlated with elevated SRC activity and tyrosine phosphorylation of CD95. We employed a recently constructed serotype 5/3 adenovirus, which is more effective than a serotype 5 virus in delivering mda- 7/IL-24 to renal carcinoma cells and which conditionally replicates (CR) in tumor cells expressing MDA-7/IL-24 by virtue of placing the adenoviral E1A gene under the control of the cancer-specific promoter progression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; CRAd.5/3-mda-7, Ad.5/3-CTV), to define efficacy in renal carcinoma cells. Ad.5/3-CTV decreased the growth of renal carcinoma tumors to a significantly greater extent than did a non-replicative virus Ad.5/3-mda-7. In contralateral uninfected renal carcinoma tumors Ad.5/3-CTV also decreased the growth of tumors to a greater extent than did Ad.5/3-mda-7. In summation, our data demonstrates that HDACIs enhance MDA-7/IL-24-mediated toxicity and tumor specific adenoviral delivery and viral replication of mda-7/IL-24 is an effective pre-clinical renal carcinoma therapeutic.

Histone deacetylase inhibitors interact with melanoma differentiation associated-7/interleukin-24 to kill primary human glioblastoma cells.

We presently demonstrate that histone deacetylase inhibitors (HDACIs) enhance toxicity of melanoma differentiation-associated gene-7/interleukin 24 (mda-7/IL-24) in invasive primary human glioblastoma multiforme (GBM) cells. Additionally, a method is described to augment the efficacy of adenoviral delivery of mda-7/IL-24 in these cells. HDACIs synergized with melanoma differentiation-associated (MDA)-7/IL-24 killing GBM cells. Enhanced lethality correlated with increased autophagy that was dependent on the expression of ceramide synthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation of reactive oxygen species and Ca(2+). Quenching of reactive oxygen species and Ca(2+) blocked HDACI and MDA-7/IL-24 killing. In vivo MDA-7/IL-24 prolonged the survival of animals carrying orthotopic tumors, and HDACIs enhanced survival further. A serotype 5/3 adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than a serotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus that conditionally replicates in tumor cells expressing MDA-7/IL-24, in which the adenoviral early region 1A (E1A) gene was driven by the cancer-specific promoter progression elevated gene-3 [Ad.5/3 (INGN 241)-PEG-E1A-mda-7; also called Ad.5/3-CTV (cancer terminator virus)]. Ad.5/3-CTV increased the survival of mice carrying GBM tumors to a significantly greater extent than did a nonreplicative virus Ad.5/3-mda-7. Ad.5/3-CTV exhibited no toxicity in the brains of Syrian hamsters. Collectively our data demonstrate that HDACIs enhance MDA-7/IL-24 lethality, and adenoviral delivery of mda-7/IL-24 combined with tumor-specific viral replication is an effective preclinical GBM therapeutic.

OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2.

BACKGROUND: Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies. METHODS: In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays. RESULTS: Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine5¹ and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser5¹Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to "rescue" the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser5¹Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment. CONCLUSIONS: These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.

hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, which are regulated by RNA trans-factors associated with exon 3 of caspase-9 pre-mRNA (C9/E3). In this study, we identified hnRNP U as an RNA trans-factor associated with C9/E3. Down-regulation of hnRNP U led to a decrease in the caspase-9a/9b mRNA ratio, demonstrating a novel enhancing function. Importantly, hnRNP U bound specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interfered with hnRNP U binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Taken together, these findings show that hnRNP U competes with hnRNP L for binding to C9/E3 to enhance the inclusion of the four-exon cassette, and this splice-enhancing function is blocked by the AKT pathway via phosphorylation of hnRNP L.

Challenges in the Treatment of Triple Negative and HER2-Overexpressing Breast Cancer.

While the 5-year survival rate of breast cancer is at an all-time high of 90%, this disease remains the second most common cause of cancer-related death, surpassed only by lung cancer in the US. The reasons for this discrepancy stem from cancer subtypes which become resistant to current therapies. These subtypes: "Triple negative" and ErbB2-overexpressing, are discussed in this review.