Rachel W. Goehe

Alternative Splicing of Caspase 9 Is Modulated by the Phosphoinositide 3-Kinase/Akt Pathway via Phosphorylation of SRp30a

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four-exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathologic classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, epidermal growth factor receptor (EGFR) overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of phosphoinositide 3-kinase or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227, and 234. Taken together, our findings show that oncogenic factors activating the phosphoinositide 3-kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.

hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

The Autophagy-Senescence Connection in Chemotherapy: Must Tumor Cells (Self) Eat Before They Sleep?

Exposure of MCF-7 breast tumor cells or HCT-116 colon carcinoma cells to clinically relevant concentrations of doxorubicin (Adriamycin; Farmitalia Research Laboratories, Milan, Italy) or camptothecin results in both autophagy and senescence. To determine whether autophagy is required for chemotherapy-induced senescence, reactive oxygen generation induced by Adriamycin was suppressed by N-acetyl cysteine and glutathione, and the induction of ataxia telangiectasia mutated, p53, and p21 was modulated pharmacologically and/or genetically. In all cases, autophagy and senescence were collaterally suppressed. The close association between autophagy and senescence indicated by these experiments reflects their collateral regulation via common signaling pathways. The potential relationship between autophagy and senescence was further examined through pharmacologic inhibition of autophagy with chloroquine and 3-methyl-adenine and genetic ablation of the autophagy-related genes ATG5 and ATG7. However, inhibition of autophagy by pharmacological and genetic approaches could not entirely abrogate the senescence response, which was only reduced and/or delayed. Taken together, our findings suggest that autophagy and senescence tend to occur in parallel, and furthermore that autophagy accelerates the development of the senescent phenotype. However, these responses are not inexorably linked or interdependent, as senescence can occur when autophagy is abrogated.

SRSF1 Regulates the Alternative Splicing of Caspase 9 Via A Novel Intronic Splicing Enhancer Affecting the Chemotherapeutic Sensitivity of Non-Small Cell Lung Cancer Cells

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed 13 possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the 4 exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non–small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the IC50 of these chemotherapeutic drugs. Finally, these studies showed that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we showed that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs. Mol Cancer Res; 9(7); 889–900. ©2011 AACR.

hnRNP U Enhances Caspase-9 Splicing and Is Modulated by AKT-dependent Phosphorylation of hnRNP L

Background: Two splice variants of caspase-9 can be generated by the inclusion/exclusion of the exon 3,4,5,6 cassette. Results: hnRNP U is an enhancer of this exonic cassette and is opposed by phosphorylation of hnRNP L via the AKT pathway. Conclusion: hnRNP U promotes the exon cassette inclusion to form caspase-9a. Significance: Understanding the regulation of caspase-9 alternative splicing is important for the treatment of lung cancer. 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.

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo. Methods The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment. Results In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin. Conclusion This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.

Photocaged permeability: a new strategy for controlled drug release

Light is used to release a drug from a cell impermeable small molecule, uncloaking its cytotoxic effect on cancer cells.

Autophagy and radiosensitization in cancer.

Autophagy is a natural self-degradative process by which cells eliminate misfolded proteins and damaged organelles. Autophagy has been shown to have multiple functions in tumor cells that may be dependent on the tumor type and the treatment conditions. Autophagy can have a cytoprotective role and be thought of as a survival mechanism or be cytotoxic in nature and mediate cell death. Radiation, one of the primary treatments for many different types of cancer, almost uniformly promotes autophagy in tumor cells. While autophagy produced in response to radiation is often considered to be cytoprotective, radiation-induced autophagy has also been shown to mediate susceptibility to radiation. This review addresses the complexity of autophagy in response to radiation treatment in three different cancer models, specifically lung cancer, breast cancer and glioblastoma. A deeper understanding of the different roles played by autophagy in response to radiation should facilitate the development of approaches for enhancing the therapeutic utility of radiation by providing strategies for combination treatment with unique radiosensitizers as well as preventing the initiation of strategies which are likely to attenuate the effectiveness of radiation therapy.

Abstract 4652: The autophagy-senescence connection in chemotherapy of breast tumor cells; senescence accelerated by autophagy but not dependent on autophagy

Introduction: Previous studies from our laboratory have established the capacity of Adriamycin to promote accelerated senescence in MCF-7 cells, while a number of studies have indicated that Adriamycin promotes autophagy. Although senescence and autophagy are considered to be two distinct cellular events in response to genotoxic stress, recent reports have suggested that the two are functionally intertwined. Thus, the current work was designed to determine whether autophagy and senescence were related in response to treatment with Adriamycin (ADR) and Campthothecin (CPT) in MCF-7 cells in vitro. Experimental Procedure: Autophagy induction was measured by acridine orange staining/quantification by flow cytometry and RFP-LC3; autophagic flux was based on p62 western immunoblotting. Autophagy was inhibited pharmacologically using 5µM Chloroquine or 5mM 3-MA and genetically using shRNA against ATG5 and ATG7. Senescence was measured by β-galactosidase staining and C12FDG fluorescence by flow cytometry. To block senescence, shRNA against p21 and 53 were used. The senescence associated markers p21, pRb, and p53 were measured by western immunoblotting. To evaluate common signaling events, 20µM KU55933 or 2mM caffeine were used to downregulate ATM and 20µM N-acetyl cysteine or 20µM Glutathione were used for scavenging ROS. Results: Both ADR and CPT collaterally induced autophagy and senescence in a time-dependent manner. Downregulation of ATM by pharmacological inhibition or genetic ablation collaterally blocked both ADR-induced autophagy and senescence. Suppression of ROS generation collaterally interfered with ADR-induced senescence and autophagy. Moreover, shRNA against either p53 or p21 resulted in a marked reduction in ADR-induced autophagy. In contrast, autophagy blockade with chloroquine, 3-MA, or shRNA against ATG5 and ATG7 only delayed senescence. Finally, tissue and protein analysis from a 4T1 breast tumor model showed that both autophagy and senescence co-exist in vivo in response to ADR. Conclusions: Treatment of MCF-7 cells with either ADR or CPT induced both autophagy and senescence. Interference with ROS generation, ATM activation and induction of p53 or p21 suppressed both autophagy and senescence. However, these observations may indicate only that both responses are mediated by common DNA-damage induced signaling pathways. When autophagy was blocked either pharmacologically or genetically, senescence was temporally delayed, but the overall extent of senescence induced by ADR or CPT was not attenuated. Consequently, although autophagy appears to accelerate and facilitate the senescence process, it is clear that senescence can occur independently of autophagy. Overall, this study provides new insights into the role of autophagy in the senescence process and the signaling events that appear to contribute to both responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4652. doi:1538-7445.AM2012-4652

Autophagy, Senescence, and Apoptosis

This chapter presents methods for interrogating the involvement of p53 in signaling to apoptosis, autophagy, and senescence. The well-known association of p53 with the stress response to chemotherapy and radiation is the basis for presenting these approaches. The development of quantitative and efficient in vitro assays has enabled researchers to overcome the limitations of previous methodologies. This chapter provides up-to-date procedures relating to the molecular networks in which the p53 protein has been shown to play a central role that allows damaged cells either to adapt to stress (autophagy and/or senescence) or to progress towards programmed cell death (apoptosis).