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Molecular and Cellular Pathobiology
Alternative Splicing of Caspase 9 Is Modulated by the Phosphoinositide 3-Kinase/Akt Pathway via Phosphorylation of SRp30a
Jacqueline C. Shultz, Rachel W. Goehe, D. Shanaka Wijesinghe, Charuta Murudkar, Amy J. Hawkins, Jerry W. Shay, John D. Minna and Charles E. Chalfant
Jacqueline C. Shultz
Rachel W. Goehe
D. Shanaka Wijesinghe
Charuta Murudkar
Amy J. Hawkins
Jerry W. Shay
John D. Minna
Charles E. Chalfant
DOI: 10.1158/0008-5472.CAN-10-1545 Published November 2010
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. Cancer Res; 70(22); 9185–96. ©2010 AACR.
Non–small cell lung cancer (NSCLC) accounts for >80% of all lung cancers diagnosed. Thus, there is an urgent need for further understanding of the molecular mechanisms of NSCLC, thereby defining new molecular targets for the treatment of this disease (1). In NSCLC, inappropriate activation of the phosphoinositide 3-kinase (PI3K) pathway by direct oncogenic mutation or from a K-Ras or epidermal growth factor receptor (EGFR) mutation has been associated with the development of NSCLC (2–4). A critical component of the PI3K nexus is the proto-oncogene Akt, a serine/threonine kinase. Akt is activated by recruitment to membranes through direct contact of its PH-domain with PtdIns (3,4,5)P3 and subsequent phosphorylation on two key residues, threonine 308 and serine 473. Previously, several laboratories reported that Akt is constitutively active in NSCLC, and experimental models have shown that Akt activation contributes to the resistance of NSCLC to chemotherapy.
An important player in the contrasting signaling cascade of apoptosis, with roles in both the sensitivity of NSCLC to chemotherapy and radiation, is Casp9. Casp9 is a member of the caspase family of proteases and is activated upon the formation of trimeric complex with cytochrome c and APAF-1, termed the apoptosome. Casp9 has roles in both the response of cells to undergo chemotherapy-induced cell death and the suppression of oncogenic transformation (5–15). The CASP9 gene produces two antagonistic isoforms, the proapoptotic Casp9a and the prosurvival Casp9b, via the inclusion/exclusion of an exon 3, 4, 5, 6 cassette (7, 12). The Casp9b isoform (exon exclusion) lacks catalytic activity while retaining key interacting domains (e.g., CARD; refs. 7, 12). Casp9b acts as an endogenous inhibitor of Casp9a by competing with the full-length Casp9a for binding to the apoptosome (7, 12). Casp9b has also been surmised to directly interact with Casp9a blocking the autoproteolysis of the enzyme (7). In this study, Casp9 splicing was shown to be dysregulated in NSCLC tumors and cell lines, and regulated by the PI3K/Akt pathway. Furthermore, this study shows that Akt exerts its effects via the phospho-status of the RNA trans-factor SRp30a.
A549, H2347, H358, H226, H2170, H596, H460, H1792, H1299, H520, H1703, H2030, H838, HCC827, and H292 cells were obtained from the American Type Culture Collection and were grown in 50% RPMI 1640 and 50% DMEM supplemented with L-glutamine, 100 units/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate. NHBE cells, from Clonetics (Cambrex Bio Science), were maintained in basal bronchial epithelial growth media (BEGM) supplemented with SingleQuot Kits-growth factors, cytokines, and supplements (Cambrex). HBEC-3KTs, HBEC-3KT K-RasV12, wild-type EGFR, and mutant EGFR HBEC-3KT cell lines were previously created and characterized (16). HBEC3-KT (E746-A750 del EGFR, K-RasV12) cell lines stably expressing control or Casp9b short hairpin RNA (shRNA) were created using lentivirus followed by selection with 20 ug/mL hygromycin (Lentigen). To generate A549, H838, and HCC827 cell lines stably expressing Casp9b cDNA, 1.5 × 105 cells were transfected with pcDNA3.1(-)/Casp9b using Effectene followed by selection with zeocin. For all comparison studies between cell lines, cells were plated in keratinocyte serum-free medium without supplements overnight prior to analysis.
Quantitative reverse transcriptase-PCR
Competitive.
Total RNA (1 μg) was reverse-transcribed using Superscript III and oligo (dT) and analyzed for Casp9, Bcl-x, caspase 8, and caspase 2 splice variants as previously described (17).
Real-time.
Total RNA was used for real-time PCR for Casp9a, Casp9b, and 18s using TaqMan PCR master mix and the Applied Biosystems 7500 Real-Time PCR System. Casp9a and 18s Q-PCR primers were ID numbers Hs00154261_m1 and Hs99999901_s1. The Q-PCR primers for Casp9b were 5-TTTGGTGATGTCGGAGCAGA-3′ (forward) and 5′-GATTTGGTGATGTCGGAGCA-3′ (reverse) and 5′-TTCCCCTGAAGACGAGTCCCCTGG-3′ (probe).
Inhibitor studies/adenoviral infection
Cells (1.2 × 105) were infected two hours with constitutively active Akt2, PTEN, or Null adenovirus (50MOI). After 48 hours, total RNA and protein were isolated.
Clonogenic and soft agar assays
For clonogenic assays, 150 viable cells were seeded into 6-well tissue culture dishes with complete growth media. Two hours postplating, cells were treated with erlonitib. After 24 hours, cells were rinsed and placed in complete media for 12 days. Colonies were counted following fixation with methanol and staining (0.1% crystal violet). For soft agar assays, cells (103) were prepared as previously described (16).
Phosphorylation assays
Twenty-four hours after transfection, cells were scraped into 150 μL of NP-40 lysis buffer [50 mmol/L Tris-HCL (pH 8.0), 150 mmol/L NaCl, 1% NP-40,100 ug of phenylmethyl-sulfonyl fluoride/mL, and protease inhibitor cocktail]. The lysates were incubated with 500 U of alkaline phosphatase for 1 hour at 37°C. Wild-type and mutant SRp30a proteins were detected using anti-T7 tag antibody.
Western immunoblotting
Immunoblotting was accomplished as previously described (18) using primary antibodies, anti-Casp9 (Assay Designs), anti-Akt1 (Santa Cruz), anti-Akt2 (Santa Cruz), anti-Akt phospho-S473 (Cell Signaling), anti-Akt pan (Cell Signaling), anti-ERK1/2 (Santa Cruz), anti-ERK1/2 phospho-Thr202/204 (Cell Signaling), anti-PDK1 (Santa Cruz), anti-T7 tag (Novagen), and anti-β-actin (Sigma-Aldrich). Secondary antibodies were horseradish peroxidase–conjugated goat anti-mouse or anti-rabbit (Sigma-Aldrich).
Transfections
The mammalian expression vectors pCGT7-SRp30a-WT and SRp30a-RD have been previously described (19). To generate SRp30a phospho-mutants, site-directed mutagenesis was performed using QuikChange XL Site-Directed Mutagenesis protocol. To analyze Casp9 splicing, 1.5 × 104 cells were transfected with 0.5 μg of pCGT7-SRp30a ± 0.2 μg of the Casp9 minigene using Effectene as previously described (17). After 24 hours, RNA and protein were isolated. For downregulation of Akt1, Akt2, and PDK1, cell lines were transfected with either Akt1, Akt2, or PDK1 SMARTpool multiplex siRNA or control siRNA (Dharmacon) using Dharmafect 1 as previously described (18). After 48 hours, RNA and protein were isolated.
When appropriate, the data are presented as mean ± SE. Data points were compared using a two-tailed, unpaired Student's t test, and the P values were calculated. P values < 0.05 were considered significant.
Casp9 RNA splicing is dysregulated in NSCLC tumors and cell lines
In this study, we examined the hypothesis that Casp9 RNA splicing was dysregulated in all pathologies of NSCLC. Utilizing total RNA from pathologist-verified human NSCLC samples, quantitative/competitive reverse transcriptase-PCR (RT-PCR) analysis was performed to determine the degree of dysregulation in the Casp9a/9b ratio as compared with matched, normal lung tissue controls (Supplementary Table S1; ref. 17). Tumor samples were categorized into three separate groups: normal, a Casp9a/9b mRNA ratio of >3.3; moderately dysregulated, a Casp9a/9b mRNA ratio of 2.3:3.3; and highly dysregulated, a Casp9a/9b mRNA ratio <2.3 (Fig. 1A and B). The normal group corresponds to the normal ratio of Casp9a/9b mRNA observed in nontransformed cells; the moderately dysregulated group corresponds to a ratio of Casp9a/9b reported to have a significant, but minor effect on Casp9 activity (15, 20); and the highly dysregulated group corresponds to a ratio of Casp9a/9b reported to significantly reduce Casp9 activity and inhibit the association of Casp9a with APAF-1 (15, 17, 20). Analysis (18, 21–23) of Casp9 splice variants showed that 36% of NSCLCs examined presented a moderately dysregulated Casp9a/9b mRNA ratio (n = 149). Importantly, 42% of tumors showed a >50% decrease in the Casp9a/9b ratio. Thus, the ratio of Casp9a/9b mRNA is significantly lower in a high percentage of NSCLC tumors irrespective of NSCLC subtype.
The Casp9a/9b mRNA ratio is dysregulated in NSCLC tumors and transformed lung epithelial cells. cDNAs from pathologist-verified lung adenocarcinomas, and squamous and large cell carcinomas (Origene) underwent quantitative/competitive PCR for expression of Casp9 splice variants. A, representation of matched pair analysis used for the degree of Casp9a/9b dysregulation in NSCLC tumors. N, normal; T, tumor. B, RT-PCR analysis of Casp9 splice variants showing 36% of NSCLC tumors are moderately dysregulated (C9a/9b ratio of 2.2:3.3) and 42% of NSCLC tumors are highly dysregulated (C9a/9b ratio < 2.2; n = 149). C, RT-PCR analysis of Casp9 splice variants from NSCLC and HBEC-3KT cells. *, dysregulated Casp9a/9b mRNA ratio; **, moderately dysregulated Casp9a/9b mRNA ratio. D, in parallel, protein expression of Casp9 splice variants.
We then examined a pure population of nontransformed lung epithelial cells, specifically primary human bronchial epithelial cells (NHBE) and immortalized HBEC-3KT cells, for the ratio of Casp9a/9b in comparison with the transformed lung epithelial cell lines A549, H838, H2347, H358, H2030, H226, H2170, H596, H1792, H1299, H520, H1703, and H292 (Supplementary Table S2). HBEC-3KT cells presented with a normal Casp9a/9b ratio of 4.02 ± 0.15, as did NHBE cells (4.15 ± 0.23; Fig. 1C). In contrast, 8 of 11 transformed lung epithelial cell lines grown under the exact same culture conditions showed a significant decrease in the Casp9a/9b mRNA ratio. Importantly, the disproportionate ratio of Casp9a/9b mRNA observed in the transformed lung epithelial cell lines translated to a disproportionate ratio of Casp9a/9b protein expression (Fig. 1D). We further validated the decrease in the Casp9a/9b mRNA ratio of A549s in comparison with HBEC-3KTs via quantitative PCR (Q-PCR; Supplementary Fig. S1), reconfirming the quantitative nature of the assay as also previously shown by ribonuclease protection assay (21). These data indicate that a significant portion of NSCLC tumors and transformed lung epithelial cells show severe dysregulation of the alternative splicing of Casp9 to favor a prosurvival/pro-oncogenic phenotype.
The EGF pathway regulates Casp9 RNA splicing in a pro-oncogenic fashion
In essentially all epithelial cancers, including NSCLC, one or more members of the family of EGFR genes are either overexpressed or mutated (4). As a large percentage of NSCLC tumors and cell lines showed a dysregulated ratio of Casp9a/9b, we next examined whether this common oncogene in NSCLC affected Casp9 RNA splicing in HBEC-3KT cells. Whereas low expression of K-RasV12 in HBEC-3KT cells (16) had no discernable effect on the ratio of Casp9a/9b mRNA, the overexpression of wild-type EGFR, the expression of L858R mutation in EGFR, and the expression of the del E746-A750 EGFR mutant induced a significant reduction in the Casp9a/9b ratio (Fig. 2A). These HBEC-3KT cell lines expressing either wild-type or mutant EGFR have previously been reported to exhibit enhanced phosphorylation of EGFR in the absence of EGF, in contrast to HBEC-3KTs expressing K-RasV12 (16). We also reconfirmed these previous findings showing increased phosphorylation of AKT in HBEC-3KT cells expressing wild-type or mutant EGFR, as well as showing an increase in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in HBEC-3KT cells expressing K-RasV12 (Supplementary Fig. S2A, B). Importantly, the del E746-A750 mutation of EGFR induced the most significant decrease in the Casp9a/9b ratio correlating with the ability of this EGFR mutant to induce anchorage-independent growth (AIG; ref. 16). Therefore, we hypothesized that increased expression of Casp9b was a major mechanism in conferring AIG of HBEC-3KT ΔE746-A750 del cells. To test this hypothesis, an E746-A750 del EGFR clonal cell line (HBEC-3KT Δ746/C9b-dr) stably expressing Casp9b shRNA was produced. The expression of Casp9b in the HBEC-3KT Δ746/C9b-dr clonal cell line was reduced to normal immunoreactive levels, and presented with a normal Casp9a/Casp9b ratio (Fig. 2B). Importantly, the HBEC-3KT Δ746/C9b-dr cells showed a complete loss of AIG compared with E746-A750 del EGFR cells stably expressing control shRNA (Fig. 2C). These effects did not require stable expression and were not due to integration artifacts as short-term/transient downregulation of Casp9b also inhibited the AIG of E746-A750 del EGFR cells (Fig. 2D and E). Furthermore, K-RasV12-expressing HBEC-3KT cells with Casp9b downregulated exhibited no difference in their AIG capacity, showing specificity for oncogenic EGFR (Supplementary Fig. S3). Thus, the distal mechanism of Casp9 splicing plays a major and specific role in the ability of EGFR signaling to confer AIG. The significance of this finding stems from the knowledge that EGFR mutation occurs in a large percentage of NSCLCs, roughly 5% to 10%, and overexpressed EGFR and its ligands occur in approximately 70% of NSCLCs. Interestingly, EGFR mutation/overexpression is also considered an early event in NSCLC because the mutation is also found in normal epithelial cells prior to metaplasia and adenoma formation, rationalizing the hypothesis that the alternative splicing of Casp9 plays a role in early events leading to the formation of NSCLC.
EGFR regulates Casp9 RNA splicing. A, RT-PCR analysis of Casp9 splice variants from vector control- and K-RasV12-expressing HBEC-3KT cells, and wild-type EGFR-, L858R EGFR-, and E746-A750 del EGFR-expressing HBEC-3KT cells (16). B and D, characterization of HBEC3-KT Δ746 cells stably (B) and transiently (D) expressing Casp9b or control shRNA by RT-PCR and Western blot analysis. C and E, colony formation assay in soft agar of HBEC-3KT Δ746 cells stably (C) and transiently (E) expressing Casp9b or control shRNA. Data are depicted as mean ± SE represented as percent control.
Modulation of Casp9 RNA splicing regulates the ability of erlotinib to inhibit the colony formation of NSCLC cells
Based on the above data, we were prompted to investigate the effect of erlotinib, a clinically relevant inhibitor of the human EGFR, on the Casp9a/9b ratio. Treatment of A549, H838, H460, and HCC827 cells with erlotinib led to a dose-dependent increase in the Casp9a/9b ratio (Fig. 3A). We further validated the increase in the Casp9a/9b ratio via Q-PCR (Supplementary Fig. S4A and B). Additionally, the effectiveness of erlotinib treatment was shown by loss of Akt phosphorylation (Supplementary Fig. S4C). Next, the effect of lowering the Casp9a/9b ratio on the sensitivity of A549 cells to erlotinib was examined. Low ectopic expression of Casp9b dramatically inhibited the ability of erlotinib to suppress cell survival (e.g., induced a resistant phenotype; Fig. 3B). In contrast, downregulation of Casp9b sensitized A549 cells to erlotinib as shown by a dramatic reduction in the IC50 (14.9 μmol/L for control shRNA to 4.8 μmol/L for C9b shRNA; Fig. 3B). To determine translatability, the effect of lowering the Casp9a/9b ratio on the sensitivity of H838 and HCC827 cells to erlotinib was also examined. H838 cells ectopically expressing Casp9b cDNA significantly inhibited the ability of erlotinib to suppress cell survival increasing the IC50 (8.5 μmol/L for vector control to 22.2 μmol/L for C9b cDNA; Fig. 3C). Conversely, downregulation of Casp9b sensitized H838s to erlotinib, reducing the IC50 to 2.9 μmol/L for C9b shRNA cells (Fig. 3C). Additionally, these same effects translated to HCC827 cells, a cell line that has been reported to be highly sensitive to erlotinib-induced growth inhibition (24). Specifically, HCC827 cells ectopically expressing Casp9b cDNA showed an increase in the IC50 of erlotinib (22.2 nmol/L for vector control to 89.9 nmol/L for C9b cDNA; Fig. 3D). Conversely, downregulation of Casp9b reduced the IC50 to 11.7 nmol/L for C9b shRNA cells (Fig. 3D). Thus, Casp9 RNA splicing plays a major role in the sensitivity of NSCLC cells to erlotinib. As erlotinib has shown promise in clinical trials, significantly improving the survival rate of NSCLC patients (25, 26), examining the alternative splicing of Casp9 may have future predictive/prognostic value for a subset of patients or allow for determination of erlotinib responsiveness in NSCLC tumors.
Casp9 RNA splicing regulates the ability of erlotinib to inhibit colony formation. A, RT-PCR analysis of Casp9 splice variants from A549, H838, H460, and HCC827 cells treated with erlotinib. Clonogenic plate assays of A549 cells (B), H838 cells (C), and HCC827 cells (D) expressing ectopic Casp9b cDNA, vector control (vec con), Casp9b shRNA, or control (con) shRNA after 24 hours in the presence of erlotinib. Data are mean ± SE represented as percent control.
The PI3K/PDK1/Akt pathway regulates Casp9 RNA splicing
We next hypothesized that a major mitogenic signaling pathway activated by EGFR regulates Casp9 RNA splicing to favor the production of Casp9b. To investigate this hypothesis, mitogenic pathways were examined for effects on the ratio of Casp9a/9b utilizing small-molecule inhibitors at doses/times reported in the scientific literature for A549 cells (27–33). Only treatment with the PI3K inhibitor LY 294002 (50 μmol/L) resulted in a significant increase in the ratio of Casp9a/9b, compared with the inactive, structurally related compound LY303511 (Fig. 4A, Supplementary Table S3). Specifically, the Casp9a/9b ratio increased from 2.10 ± 0.18 for control samples to 3.91 ± 0.08 for inhibitor-treated samples (P < 0.0008; n = 6). The inhibition of PI3K effectively returned the Casp9a/9b ratio to the ratio observed in nontransformed lung epithelial cells. Importantly, this effect on the Casp9a/9b ratio translated to the protein level (Fig. 4A).
The PI3K/PDK1/Akt pathway regulates Casp9 RNA splicing. RT-PCR analysis and Western immunoblot of Casp9 splice variants from A549 cells (A) and H838, H2030, H358, H292, and H520 cells (B) treated with either LY294002 (50 μmol/L) or LY303511 (50 μmol/L). C, D, and E, RT-PCR analysis of Casp9 splice variants and the corresponding Casp9a/9b mRNA ratios from A549 cells treated with 0.1% DMSO control and the Akt inhibitor Akt VIII (25 μmol/L; C), SH-5 (25 μmol/L; D), or OSU03012 (10 μmol/L; E). F, RT-PCR analysis of Casp9 splice variants and Western immunoblot analysis of PDK1 from A549s transfected with scrambled siRNA (100 nmol/L) or PDK1 siRNA (100 nmol/L). G, A549 cells were treated with the Akt inhibitor Akt VIII (25 μmol/L) and analyzed by RT-PCR for Bcl-x, caspase 8, and caspase 2 splice variants. Data are expressed as means ± SE.
To determine translatability, H838, H2030, H358, H292, and H520 cells were also treated with LY294002 (Fig. 4B). Again, the Casp9a/9b ratio was dramatically increased (H838 cells from 1.24 ± 0.05 to 3.29 ± 0.19; H2030 cells from 2.30 ± 0.08 to 3.74 ± 0.08; H358 cells from 3.11 ± 0.09 to 4.13 ± 0.12; H292 cells from 2.01 ± 0.09 to 4.11 ± 0.11; and H520 cells from 2.35 ± 0.08 to 3.91 ± 0.13). Therefore, the ability of the PI3K pathway to regulate the alternative splicing of Casp9 translates to multiple NSCLC cell lines.
Akt/PKB, SGK, PKCξ, and PKCδ are downstream of PI3K (34). To investigate the downstream effector of PI3K responsible for regulating Casp9 RNA splicing, small-molecule inhibitors in conjunction with RNA interference technology was employed. Treatment of A549 cells with the PKC inhibitors GÖ6976 (10 μmol/L) and GÖ6983 (10 μmol/L; Supplementary Fig. S5A and B, Supplementary Table S3) resulted in no significant change in the Casp9a/b ratio. Conversely, treatment of A549 cells with the Akt inhibitor Akt VIII (25 μmol/L) and the phosphatidylinositol analog SH-5 (10 μmol/L) resulted in an increased Casp9a/9b ratio to the same extent as PI3K inhibition, from 2.10 ± 0.11 for DMSO to 4.03 ± 0.13 for Akt VIII (n = 6; P < 0.01) and from 2.10 ± 0.08 for DMSO to 3.73 ± 0.13 for SH-5 (n = 4; P < 0.01; Fig. 4C and D). In addition, treatment of cells with the PDK1 inhibitor OSU03012 (10 μmol/L) or siRNA downregulation of PDK1 (Fig. 4E and F) resulted in a significantly increased Casp9a/9b ratio, from 2.25 ± 0.13 for control to 4.05 ± 0.22 for OSU03012 (n = 4; P < 0.01) and from 2.13 ± 0.09 for siRNA controls to 3.78 ± 0.21 for siPDK1 (n = 4; P < 0.01). To verify appropriate inhibition of downstream signaling pathways, the phosphorylation status of Akt was examined via Western immunoblotting (Supplementary Fig. S6A–C).
These data show that Akt regulates Casp9 RNA splicing in an antiapoptotic/prosurvival manner. This effect was specific for Casp9 RNA splicing, and not a generalized effect on constitutive RNA splicing, as no effect on the alternative splicing of caspase 8, caspase 2, and Bcl-x pre-mRNA was observed with Akt VIII treatment (Fig. 4G). Furthermore, cotreatment of A549 cells with both Akt VIII and LY294002 could not further increase the Casp9a/9b ratio, showing a linear pathway with Akt as the downstream effector of PI3K (Supplementary Fig. S7).
To examine the Akt isoform responsible for regulating the alternative splicing, siRNA was again utilized. Downregulation of Akt1 using multiplex siRNA resulted in no significant change in the Casp9a/b ratio (P > 0.34; Fig. 5A). In contrast, downregulation of Akt2 resulted in a dramatic increase in the Casp9a/b ratio from 2.10 ± 0.05 to 4.09 ± 0.25 (P < 0.01; n = 5; Fig. 5B). Again, this effect translated to the protein level (Fig. 5C).
Akt regulates Casp9 RNA splicing via PIP3 membrane interactions. A and B, RT-PCR analysis of Casp9 splice variants from A549 cells transfected with control siRNA or Akt1 siRNA (A), and control siRNA or Akt2 siRNA (B). C, Western blot analysis for Casp9. D and E, RT-PCR analysis of Casp9 splice variants from A549s infected with null or constitutively active Akt2 (D), or null and PTEN adenovirus (E). F, H2030, H358, H292, H520, and HCC827 cells were treated with 0.1% DMSO or Akt inhibitor VIII (25 μmol/L) for 24 hours. Total RNA was isolated and RT-PCR performed for Casp9 splice variants. G, RT-PCR analysis of Casp9 splice variants from H358s transfected with control siRNA or Akt1 siRNA. Data are expressed as means ± SE.
To further validate a role for Akt2, expression of constitutively active Akt2 (always membrane bound via myristoylation) induced a significant decrease in the Casp9a/9b ratio (Fig. 5D). Furthermore, ectopic expression of PTEN induced the contrasting effect (Fig. 5E). Therefore, Akt2 regulates Casp9 RNA splicing via PIP3 membrane interactions in A549 cells, and also requires PDK1 phosphorylation.
To determine translatability, H2030, H292, H358, H520, and HCC827 cells were treated with Akt VIII. Indeed, all cell lines also showed a dramatic increase in the Casp9a/9b ratio (Fig. 5F). Additionally, Akt1 and Akt2 were downregulated in H358 cells to determine the Akt isoform responsible for regulating Casp9 RNA splicing. In contrast to A549s, downregulation of Akt1, but not Akt2, induced a significant increase in the Casp9a/9b ratio (Fig. 5G). Therefore, multiple Akt isoforms have the potential to regulate Casp9 RNA splicing in NSCLC cells.
Lastly, as with the NSCLC cell lines, the ability of del 746-750 EGFR mutation to induce a lowering of the Casp9a/9b ratio in HBEC-3KT cells was dependent on PI3K and Akt as treatment with LY294002 and Akt VIII increased the ratio of Casp9a/9b (Fig. 6A and B). These data suggest that EGFR overexpression/mutation correlates with Akt activation affecting the alternative splicing of Casp9. We further extended these results by examining the Akt isoform responsible for regulating Casp9 RNA splicing in HBEC-3KT (del E746-A750 EGFR) cells. Interestingly, siRNA against both Akt1 and Akt2 (Fig. 6C) resulted in a significantly increased Casp9a/Casp9b ratio as compared with control siRNA, from 2.65 ± 0.05 for siRNA control to 4.01 ± 0.13 for siAkt1 (n = 3; P < 0.01) and to 3.72 ± 0.11 for siAkt2 (n = 3; P < 0.01). Therefore, multiple Akt isoforms are responsible for regulating the alternative splicing of Casp9. The finding that the Akt pathway regulates this splicing mechanism important for EGFR conferring AIG makes logical sense in relation to cellular transformation, as this pathway is found constitutively active in approximately 58% of NSCLC cell lines and tumors (35, 36). This pathway is also linked to constitutive EGFR activity, NF-κB activation, and the ability of oncogenic Ras to transform various cell types (37–39). Thus, the Akt pathway activated by EGFR is extremely important in a therapeutic sense, and this study suggests that the alternative splicing of Casp9 is a key distal mechanism in the biological role of this pathway in NSCLC development/maintenance.
Akt regulates Casp9 RNA splicing in E746-A750 del EGFR-expressing HBEC-3KT cells. A and B, RT-PCR analysis of Casp9 splice variants from E746-A750 EGFR-expressing HBEC3-KT cells treated with 50 μmol/L LY294002 or 50 μmol/L LY303511 (A), and with 0.1% DMSO or 25 μmol/L of the Akt inhibitor Akt VIII (B). C, RT-PCR analysis of Casp9 splice variants from E746-A750 EGFR-expressing HBEC3-KT cells transfected with control siRNA, Akt1 siRNA, or Akt2 siRNA. Data are expressed as means ± SE.
How Casp9b acts to drive AIG is more of an enigma. As we have shown, removal of Casp9b blocked the ability of EGFR (del E746-A750) to induce AIG. This is likely not attributed to the blockade of an initiator caspase such as Casp9a, and suggests a role in cell signaling for Casp9b. In this regard, the possibility that Casp9b acts as a signaling molecule has been reported by Latchman and coworkers (5). Specifically, this laboratory group showed that ectopic expression of Casp9b induced the activation of NF-κB irrespective of caspase activation (5). Activation of NF-κB by Casp9b expression also "fits" well with cooperation with K-Ras mutations for the induction of cellular transformation based on the findings of Ma and Baldwin (40, 41). These laboratory groups showed in several different ways that NF-κB activation enhances the ability of oncogenic ras to induce cellular transformation. Coupled with the knowledge that EGFR overexpression/mutation leads to both cooperation with oncogenic ras in cellular transformation and NF-κB activation, a role for Casp9b in these pathways important for cellular transformation is logical. Thus, Casp9b may act as a scaffolding protein to elicit downstream signaling events with roles outside the simple inactivation of Casp9a. This possibility is far from inconceivable as the initiator caspase, caspase 8, is reported to recruit cell survival factors such as PI3K subunits (42–44). Although this caspase is an initiator of extrinsic pathways of apoptosis, Casp9b may play an analogous role in survival signaling as an initiator of the intrinsic pathway of apoptosis.
The phospho-status of SRp30a modulates the effect of Akt signaling on Casp9 RNA splicing
Our laboratory reported that SRp30a was a required enhancer factor for the inclusion of the exon 3,4,5,6 cassette of Casp9 (18). Furthermore, SRp30a has been shown to be a specific target of Akt in vitro (45, 46). Therefore, we hypothesized that the phospho-status of SRp30a regulates the inclusion of the exon 3,4,5,6 cassette of Casp9, downstream of Akt activation. To investigate this hypothesis, we employed an established phospho-mimic of SRp30a, SRp30a-RD, in which the majority of serine residues in the RS domain were mutated to aspartic acid (19). Coexpression of SRp30a-RD with a functional Casp9 minigene induced a significant decrease in the Casp9a/9b ratio compared with wild-type SRp30a and empty vector controls (Fig. 7A). Importantly, expression of SRp30a-RD also induced a significant decrease in the endogenous Casp9a/9b ratio as compared with wild-type SRp30a and empty vector controls (Fig. 7B).
The phospho-status of SRp30a regulates Casp9 RNA splicing. A, B, C, and D, RT-PCR analysis of Casp9 minigene-derived transcripts (A and C) or endogenous Casp9 splice variants (B and D) from A549 cells transfected with the Casp9 minigene and SRp30a-WT or SRp30a-RD (A); SRp30a-WT or SRp30a-RD (B); and the indicated SRp30a phospho-mutants (C and D). E, F, G, and H, Western blot analysis of the phosphorylation state of SRp30a. Lysates were analyzed for the expression/migration of T7-tagged SRp30a. E, A549 cells were transfected with the indicated T7-tagged SRp30a constructs and total cell lysates were incubated in the presence of either active or denatured AP. F, HBEC-3KT and A549 cells transfected with the indicated T7-tagged SRp30a constructs. G, endogenous SRp30a from A549 cells treated with 0.1% DMSO or erlotinib (50 μmol/L) for 48 hours. Total protein lysates were incubated in the presence of either active or denatured AP. H, endogenous SRp30a from A549 cells treated with 0.1% DMSO or Akt VIII (50 μmol/L). I, RT-PCR analysis of endogenous Casp9 splice variants from A549s transfected with SRp30a-WT or SRp30a-QD ± Akt inhibitor Akt VIII (25 μmol/L). Data are expressed as means ± SE.
To determine the serine residue/residues of SRp30a required for regulating the alternative splicing of Casp9, site-directed replacement mutagenesis was utilized. Multiple serine residues in the RS domain of SRp30a contain recognized motifs for Akt phosphorylation and numerous residues have been validated by mass spectrometric analysis (Supplementary Table S4; ref. 45). These serine residues were individually mutated into aspartic acid to produce phospho-mimics (Supplementary Table S4). Coexpression of only the SRp30a-S199D, SRp30a-S201D, SRp30a-S227D, and SRp30a-S234D mutants with a functional Casp9 minigene decreased the Casp9a/9b ratio compared with wild-type SRp30a control (Fig. 7C).
We extended these results to produce a SRp30a double phospho-mutant (SRp30a-S199,201D), a SRp30a triple phospho-mutant (SRp30a-S199,201,234D), and a SRp30a quadruple (SRp30a-QD) mutant harboring serine to aspartic acid mutations at residues 199, 201, 227, and 234. Expression of SRp30a double and triple phospho-mutants with the Casp9 minigene further decreased the Casp9a/9b ratio (Supplementary Fig. S8). Ectopic expression of SRp30a-QD induced a reduction in the Casp9a/9b ratio comparable with the SRp30a-RD mutant. Conversely, a quadruple dephospho-mimic of SRp30a (SRp30a-QA) induced the opposite effect in A549 cells (Fig. 7D). These effects could not be due to localization issues as both SRp30a quadruple mutants are localized in the nucleus and were expressed in equivalent amounts (Supplementary Fig. S9).
To show that these phospho-sites are hyperphosphorylated in NSCLC, the phosphorylation status of the transiently expressed SRp30a was analyzed by comparing the electrophoretic migration profiles in the presence of either alkaline phosphatase (AP) or denatured AP. A dramatic increase in the migration of SRp30a-WT was observed after treatment with AP, indicating that this protein is phosphorylated in A549 cells. In contrast, the migration of SRp30a-QD and SRp30a-QA after treatment with AP was significantly decreased in comparison with the migration of SRp30a-WT, indicating that these proteins are phosphorylated to a significantly lesser extent (Fig. 7E). These data show that serines 199, 201, 227, and 234 exist in a phosphorylated state in A549 cells. Moreover, the electrophoretic migration profiles of A549s and HBEC3-KT cells after transfection with SRp30a-WT and SRp30a-QA indicate that SRp30a exists in a decreased phosphorylated state in nontransformed cells versus NSCLC cells (Fig. 7F).
We extended these results to determine the effect of inhibiting the EGFR/Akt pathway on the phosphorylation status of SRp30a in A549 cells. As predicted, an increase in the migration of endogenous SRp30a was observed after treatment with erlotinib (Fig. 7G), as well as after treatment with the Akt inhibitor Akt VIII (Fig. 7H). To determine whether the PI3K/Akt pathway regulates Casp9 RNA splicing in a phospho-SRp30a-dependent manner, SRp30a-QD was expressed in the presence or absence of Akt VIII. In the presence of SRp30a-QD, Akt VIII was unable to increase the ratio of the Casp9a/9b to the same extent as compared with wild-type SRp30a (Fig. 7I). Therefore, the Akt pathway regulates the alternative splicing of Casp9 at least partially via the phospho-state of SRp30a on serines 199, 201, 227, and 234.
These data solidify a role for phosphorylation of SRp30a in regulating the alternative splicing of Casp9, but also suggest additional regulating mechanisms. In this regard, our laboratory recently found that the RNA trans-factor hnRNP L acts as a repressor for the inclusion of the exon 3,4,5,6 cassette of Casp9, and its repressor activity is regulated by the phosphorylation status of Ser52 (17). Thus, we hypothesize that the EGFR/PI3K/Akt pathway may also regulate the phospho-status of hnRNP L at Ser52, suggesting a coordinated interplay between these two trans-factors in regulating the alternative splicing of Casp9. This possibility is logical, as Lynch and coworkers showed the ability of SRp30a and hnRNP L to directly compete for binding to the exon 5 regulatory sequence of CD45 and that this interplay between SRp30a and hnRNPL influences the extent of exon inclusion (47).
The phospho-state of SRp30a regulating the inclusion of the exon 3,4,5,6 cassette also fits with our previous findings that ceramide induced both the dephosphorylation of SRp30a and the inclusion of the Casp9 exon cassette. SRp30a was also required for ceramide effects on the inclusion of the exonic cassette of Casp9. Thus, the regulation of SRp30a phosphorylation and the alternative splicing of Casp9 may be a key distal point by which ceramide acts as a tumor suppressing/cell senescence agent as the ceramide signaling and PI3K/Akt pathway are well established to antagonize each other (Supplementary Fig. S10).
In conclusion, the presented study reports several major findings taking a comprehensive approach. First, the dysregulation of the alternative splicing of Casp9 toward a prosurvival phenotype was shown in NSCLC. Second, a survival/mitogenic/oncogenic pathway involving EGFR, PI3K, and Akt was shown to regulate this splicing mechanism. Lastly, the phospho-state of SRp30a was shown to regulate this distal mechanism via Akt signaling. Therefore, the present study shows a novel and key distal mechanism in NSCLC and provides new target mechanisms for the development of therapeutics to combat the cancer with the highest mortality rate.
No potential conflicts of interest were disclosed.
Grant Support: Veteran's Administration (VA Merit Review I and a Research Career Scientist Award to C.E.C.), from the National Institutes of Health (HL072925 (C.E. Chalfant), CA117950 (C.E. Chalfant), NH1C06-RR17393 (VCU), NCI Lung Cancer SPORE P50CA70907 (J.D. Minna), NRSA-T32 Fellowship (D.S. Wijesinghe), and from NASA, NNJO5HD36G (J.D. Minna).
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Revision received August 27, 2010.
Accepted September 4, 2010.
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Cancer Res November 15 2010 (70) (22) 9185-9196; DOI: 10.1158/0008-5472.CAN-10-1545
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This super moisturising formula with Hyaluronic Acid + Mint + Peppermint essential oils cleans all the hard-to-reach areas in your mouth, protects and soothes your gums and provides extremely long-lasting freshness. So, get fresh and give your mouth a good cleaning. For children below the age of 6 use small quantity. Make sure that they do not swallow. If you are using any other products containing fluoride, please consult your dentist or your doctor.


Danganronpa (ダンガンロンパ?) is a Japanese video game franchise created and developed by Spike Chunsoft (formerly Spike) for the PlayStation Portable, PlayStation Vita, iOS, Android, and home computers. The series currently consists of three main titles; two visual novel games, Danganronpa: Trigger Happy Havoc and Danganronpa 2: Goodbye Despair, and a third-person adventure spin-off, Danganronpa Another Episode: Ultra Despair Girls, along with an upcoming third visual novel, New Danganronpa V3: Minna no Koroshiai Shingakki, and multiple smartphone titles, as well as various manga, novel, and anime adaptations. The games are published in North America and Europe by NIS America. The series' name is a portmanteau of dangan (弾丸?, bullet) and ronpa (論破?, confutation).


"Obesogens", a term coined for industrial toxins present in our environment, might very well be the primary reason behind obesity.
Obesity is spreading like an epidemic, but it looks like poor diet and health routines along with lack of exercise and willpower, aren't the only ones to blame after all.
Research published in the Journal of Alternative and Complementary Medicine in 2002 has linked obesity with the rise in industrial toxins.
These toxins are harmful to human health and responsible for causing obesity, birth defects, and cancer because of their effect on the endocrine system.
Initial effects of these toxins were uncovered in the 1980s on breast cancer cells.
They promote the growth of cancer cells, and further research revealed the exposure to these chemicals also caused infertility and mutations in reproductive organs.
How Do They Cause Obesity?
Exposure to these endocrine disrupting toxins like BPA and phthalates among others has various effects on the cells that promote obesity.
It also depends on the timing of exposure as it has been found that pregnant women with exposure to these toxins, can pass it into the developing fetus.
This kind of exposure in the early development stages can set the stage for diabetes or obesity.
These toxins can harm natural health in a number of ways such as through the interaction with the brain, liver, fat cells, or pancreas.
In any case, the result is going to be obesity and diabetes, which in turn gives rise to other health conditions as well.
These obesogens, rightly termed, also encourage the body to store fat and program cells into becoming fat cells rather than muscle or bone.
They can also make the liver resistant to insulin, converting more calories into fat.
They also mess with the hormones, such as that of leptin, preventing its release so that the body does not get the signal that it is full.
This promotes overeating in individuals.
We are exposed to these obesogens in quite a number of ways.
These toxins are present in everything, from household products, canned food linings, wood furniture to paint, plastic bottles, toys, cosmetics, and others.
They are also present in herbicides, fungicides, and pesticides, all of which are used to produce the fruits and vegetables that we eat.
Eliminating each and every one of these products from our lives is impossible because these toxins are present in just about everything.
To limit your exposure to obesogens, you can start by switching to organic foods instead of mass-produced items that are sprayed with pesticides. And whenever possible, buy organic home cleaning and personal care products, too.
Research and increase your understanding of the presence and exposure to these chemicals to lower your risk of obesity and other diseases.
We regularly use a product called Cleanse For Life.
It contains over 20 natural ingredients that will help your body release toxins so you can think more clearly, lose weight, and have more energy.
If you need to lose weight I recommend starting with the Isagenix 30 Day Cleansing System. It includes Cleanse For Life in a whole program that is designed to nourish and cleanse your system so you can lose weight and get healthier.
Obesogens are toxins that can cause us to hold on to weight and eventually cause us to be unhealthy. They are here to stay so we have to find ways to reduce them in our homes and bodies.
Little changes every day will go a long way in protecting yourself and your family.
Did you already know about Obesogens? If not, how will this information change the way you think about things?
Do Environmental Toxins Affect Fertility?


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In Florida, a party can recover attorney's fees if it has a contractual or statutory basis. If a party has neither a contractual or statutory basis to recover attorney's fees, another vehicle is to serve an Offer of Judgment (also known as a Proposal for Settlement). Whether there is an argument to recover attorney's fees is an important consideration.
The Florida Deceptive and Unfair Trade Practices Act ("FDUTPA") is a consumer-protection related law designed to allow parties to sue other parties for deceptive and unfair trade or business practices. FDUTPA is discussed in more detail in http://www.floridaconstructionlegalupdates.com/actual-damages-under-floridas-deceptive-and-unfair-trade-practices-act/. There are certain circumstances when asserting a FDUTPA claim is worthwhile and should be explored such as when a party is looking for a statutory basis to recover attorney's fees.
The reason the word "may" is highlighted is because this is permissive, not mandatory, language. In other words, it is not automatic or mandatory that attorney's have to be awarded to the prevailing party, but they could (i.e., may) be awarded. This is an important distinction. However, recently, Florida decisions have indicated that attorney fees should be mandatorily awarded to the prevailing party in a FDUTPA action.
Bull Motors, supra (internal quotations omitted).
Bull Motors relied on the Florida Supreme Court's decision in Diamond Aircraft Indus., Inc. v. Horowitch, 107 So.3d 362 (Fla. 2013). In Diamond Aircraft, a plaintiff asserted a FDUTPA claim against a defendant. However, it was determined that FDUTPA did not apply because Arizona law, not Florida law, governed the case. Thus, the defendant prevailed under the plaintiff's FDUTPA claim. A question certified to the Florida Supreme Court to answer was whether FDUTPA entitled a prevailing party to attorney's fees if the court determines that FDUTPA does not apply to the case because the substantive law of another state (in this case, Arizona) applied. The Court stated that it did (or answered the question in the affirmative) holding that by a plaintiff asserting a FDUTPA claim, it exposes itself to both the benefits and potential consequences of the statute. Further, the Court expressed: "Under FDUTPA, a prevailing party is entitled to reasonable attorney's fees and costs in civil litigation arising from a violation of that act 'after judgment in the trial court and exhaustion of all appeals.'" Diamond Aircraft, supra, at 370 quoting Fla.Stat. s. 501.2105.
Hence, even though the statute contains permissive language, there is strong legal authority that would mandatorily entitle a prevailing party to recover attorney's fees. This cuts both ways. This means that a plaintiff could expose itself to attorney's fees by improperly asserting a FDUTPA claim without facts to support a deceptive or unfair trade practice or without evidence to support actual damages as provided under the statute. Plaintiffs need to be cognizant of this before asserting a FDUTPA claim.
In a nutshell, a defendant can recover its attorney's fees if it serves an Offer of Judgment and the plaintiff gets a $0 judgment against the defendant or the plaintiff gets a judgment of at least 25% less than the offer. For example, and using simple math, let's say the defendant serves an Offer of Judgment for $100,000 and the plaintiff obtains a net judgment against the defendant for $50,000. In this situation, the defendant could be entitled to its attorney's fees from the date of the Offer of Judgment and forward since the plaintiff obtained a judgment of at least 25% less than its $100,000 Offer.
And, if a plaintiff serves an Offer of Judgment, it can recover its attorney's fees if gets a net judgment of at least 25% greater than the Offer. Let's say the plaintiff serves a $100,000 Offer of Judgment and recovers a net judgment against the defendant for $150,000. In this situation, the plaintiff could be entitled to its attorney's fees from the date of the Offer of Judgment and forward since the plaintiff obtained a judgment of at least 25% greater than its $100,000 Offer.
"Courts have also held that when a plaintiff seeks both monetary and nonmonetary relief, and a party makes a general offer of settlement, section 768.79 is not applicable. The reasoning adopted in those decisions is that strict construction of the phrase "any civil action for damages" in the offer of judgment statute does not include a claim for equitable relief, or one that involves claims for both monetary and nonmonetary relief.
Diamond Aircraft, 107 So.3d at 373-74 (internal citations omitted); accord Bull Motors, supra ("The offer of judgment statute, section 768.79…does not apply to cases that, as here, involve a general offer seeking release of all claims in the case, both equitable and monetary.").
Under Florida's Deceptive and Unfair Trade Practices Act ("FDUTPA"), a party can only recover in principal what the statute refers to as "actual damages." See Fla. Stat. s. 501.211(2) ("In any action brought by a person who has suffered a loss as a result of a violation of this part, such person may recover actual damages, plus attorney's fees and court costs….") However, the statute does not define the term actual damages and, thus, parties need to analyze Florida caselaw to understand the meaning of actual damages. This is important so parties know the damages covered under FDUTPA. A claim under FDUTPA is sometimes asserted in a construction-related dispute. Sometimes, it is asserted if a party is seeking an avenue to potentially recover attorneys' fees.
Florida courts (or federal courts interpreting Florida law) have maintained that "actual damages" refer to the "difference in the market value of the product or service in the condition it was delivered and its market value in the condition in which it should have been delivered according to the contract of the parties. A notable exception to the rule may exist when the product is rendered valueless as a result of the defect-then the purchase price is the appropriate measure of damages." Rollins, Inc. v. Butland, 951 So.2d 860, 869 (Fla. 2d DCA 2006) quoting Rollins, Inc. v. Heller, 454 So.2d 580, 585 (Fla. 3d DCA 1984).
This is similar to benefit of the bargain damages – the market value of the product represented minus the market value of the product delivered. See Gastaldi v. Sunvest Resort Communities, LC, 709 F. Supp. 2d 1299, 1304 (S.D.Fla. 2010).
Based on the way Florida cases define actual damages under FDUTPA, a party needs to prove its damages in accordance with this definition by analyzing the market value of the product represented versus the market value of the product actually received / delivered.
– Gastaldi v. Sunvest Resort Communities, LC, 709 F. Supp. 2d 1299, 1307 (S.D.Fla. 2010)– measure of damages would be the difference in the market value of the condominium units with the condominium having a luxury sports club in 2009 [as represented] minus the market value of the condominium units without the condominium having a luxury sports club in 2009 [as delivered].
Notably, actual damages under FDUTPA does not include consequential-type damages (or damages other than those established by the measure provided above). See Dorestin v. Hollywood Imports, Inc., 45 So.3d 819, 824-25 (Fla. 4th DCA 2010) (FDUTPA does not allow for consequential damages or any other damages outside of actual damages); Orkin Exterminating Co., Inc. v. DelGuidice, 790 So.2d 1158, 1162 (Fla. 5th 2001) (actual damages do not include actual consequential damages). For example, lost profits or interest on payments would not be a recoverable consequential damage. See Rodriguez v. Recovery Performance & Marine, LLC, 38 So.3d 178 (Fla. 3d DCA 2010) and Siever v. BWGaskets, Inc., 669 F.Supp.2d 1286, 1294 (M.D.Fla. 2009).
Understanding actual damages in a FDUTPA claim is important prior to asserting a claim so that a party knows what is recoverable and what is not recoverable under FDUTPA. It is also important so that a party knows how to prove actual damages.


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text:
Long-running animosity escalates with arrest
By Paula J. Owen TELEGRAM & GAZETTE STAFF
The arrest of mayoral candidate Kevin A. Lynch last month after an altercation with Sgt. Kerry C. Siomos Jr. called attention to a long-running dispute between the two men.
The sergeant, in his report on the arrest, alleged that Mr. Lynch had been harassing and stalking him.
But Mr. Lynch said he has just been looking out for the welfare of the city, and was outraged that the sergeant appeared to be taking advantage of his job as a police officer to boost business at his family�s business, JR�s Pub at 14 North St.
Mr. Lynch, 41, of 48 Cedar St. said he had filed many requests with Mayor Lisa A. Wong for an internal affairs investigation of the sergeant�s activities. And after the March 23 altercation, for which Mr. Lynch faces charges of threatening to commit a crime (murder), assault and battery on a police officer, assault and battery on ambulance personnel and disorderly conduct, Mr. Lynch filed countercharges in district court against Sgt. Siomos. Mr. Lynch accuses the sergeant of assault and battery, threat to murder and being a police officer filing false charges.
Judge Timothy M. Bibaud, from the Western Worcester District Court in East Brookfield, has been given jurisdiction over the case and will oversee the hearing and future proceedings and also decide if criminal charges against Sgt. Siomos will move forward. Mr. Lynch�s pretrial hearing is May 4 in Fitchburg District Court.
Mr. Lynch�s lawyer�s request to preserve possible videotape of the incident taken from cameras outside the fire station was allowed.
Mr. Lynch�s friend David L. Gonzalez, 41, of 53 Forest St., is charged with assault and battery on a police officer stemming from the same incident, when he allegedly tried to stop police from arresting Mr. Lynch.
In his report on the incident, Sgt. Siomos said Mr. Lynch assaulted him around 8:45 p.m. outside JR�s Pub, which is across the street from the North Street fire station, when the officer walked outside to move his car.
�(Mr. Lynch) continued to assault this officer, at which time I punched subject taking him to the ground,� Sgt. Siomos� report said.
Sgt. Siomos said he told Mr. Lynch to stay on the ground, but when the mayoral candidate got up and went after Sgt. Siomos again, Firefighter P.J. Roy � the fire chief�s son � put his arms around him to restrain him. Mr. Lynch then began striking him with his elbows to get away, according to Sgt. Siomos� report.
But Mr. Lynch claims that Sgt. Siomos threw open the doors at JR�s and came after him yelling, and punched him repeatedly before he fell to the ground. When he got up, he said, Firefighter Roy restrained him while the off-duty sergeant punched him in the face several more times.
The sergeant, he said, also took his iPhone, which Mr. Gonzalez was using to try to record the incident. He said the iPhone also contained photos of cars illegally parked at JR�s on other occasions.
Mr. Lynch was taken to HealthAlliance Hospital � Leominster Campus for treatment and was released.
Mr. Lynch said that just before the altercation he had called 911 about cars illegally parked at JR�s that night � a business listed as owned by Sgt. Siomos� family.
He said that for the past year he has asked officials, including the mayor and police chief, to look into illegal parking outside the bar by patrons and off-duty police. He said he is concerned about emergency vehicles going in and out of the fire station.
He said that when he called 911 on March 23, the dispatcher told him to wait there and not call for tow trucks. Mr. Lynch claims the dispatcher then called Sgt. Siomos� cellphone to alert him that Mr. Lynch was outside JR�s. Right after that, Mr. Lynch said, the sergeant burst out of the bar and the altercation took place.
On April 15, Mr. Lynch requested an investigation into the incident with the Worcester district attorney�s office and Department of Justice.
Timothy J. Connolly, spokesman for the Worcester DA�s office, said the case is being handled by the FBI.
Police Chief Robert A. DeMoura said he could not comment on pending investigations, but did confirm that Mr. Lynch has filed many requests with Mayor Lisa A. Wong for internal affairs investigations of Sgt. Siomos.
When contacted, Ms. Wong said she had no comment.
Sgt. Siomos and Firefighter Roy did not return calls seeking comment.
Meanwhile, the legal status of the bar is in question.
Brian McNiff, spokesman for the secretary of state�s office, said JR�s Inc., 18 Putnam St., which holds the liquor license for JR�s Pub, was dissolved in 1998. Sgt. Siomos� father, Kerry C. Siomos Sr., was listed as the president and treasurer of the corporation.
William A. Judge, chairman of Fitchburg�s license commission, said the original application for the license was made by Sgt. Siomos� family when the bar was at 18 Putnam St., and commission renewals are �almost automatic.� The validity of the current license is questionable, he said.
�It is very possible over the years it has just gone through,� said Mr. Judge. �If it is a new application, we always check the corporation and who is behind it, but renewals are almost automatic. We don�t necessarily go back and look. Now that this has come up, maybe we should spot check. I really can�t give an answer �yes� or �no� if the license is valid. It is questionable that we have been renewing it all these years as JR�s Inc.�
In a related matter, he said there is no law prohibiting a police officer or his family from owning a bar.
�There is nothing that prevents police officers from owning a bar in state law or in city code, but in other communities, police chiefs have had an issue with officers owning bars,� said Mr. Judge. �The paperwork we�ve seen never listed Sgt. Siomos as the owner � just his family. The manager was always listed as his mother. I�ve heard people say the commission has given them preferential treatment, but we have never done that with anyone.�
Mr. Judge said the commission does not oversee alleged illegal parking at a bar. That is the kind of question that is handled by the police, he said.
Mr. Lynch has a three-page criminal history dating to when he lived in Boston.
He was incarcerated in 1992 for seven years for manslaughter and has several disorderly conduct convictions predating the manslaughter conviction, as well as a conviction for assault and battery on a police officer.
Mr. Lynch moved to Fitchburg in 2000. He said he plans to continue his bid for mayor. He is a self-employed carpenter.

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It's becoming clear that enterprises need more than one cloud. Hybrid enables enterprises to optimize how their business works – public cloud for elasticity and scale, multi-cloud for redundancy and choice, and on-premises for performance and privacy. However traditional data warehouses and first-generation cloud data warehouses have typically enabled one cloud choice – theirs. Cloudera Data Warehouse helps people work with verifiable data irrespective of whether it's in a public cloud, across multi-clouds, or on-premises. Cloudera delivers a hybrid cloud data warehouse that works where enterprises work, with the agility, security and governance enterprise IT needs, and the self-service analytics business people and enterprise data professionals demand.
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Companies like Uber and Airbnb are disrupting entire industries and changing the way we consume. It's time to rethink logistics with access over ownership.
Have you ever hailed an Uber rather than a taxi, booked an Airbnb rather than a hotel, or bought on eBay rather than at your local store? Then you've already participated in the sharing economy, which has also been called on-demand, peer-to-peer, collaborative – and more! – depending on who you talk to. Call it what you will, but the sharing economy is here to stay and poised to grow significantly in the coming years.
PricewaterhouseCoopers has identified five key sharing sectors with high growth potential – travel, car-sharing, finance, staffing and music/video streaming – and estimates that these sectors alone will increase the sharing economy from the US$15 billion industry it was in 2014 to a US$335 billion in 2025.
But what about the sharing economy in the logistics industry? The notion of sharing is actually not new to logistics. In fact, DHL leveraged the idea in its early days, offering free plane tickets to private travelers in exchange for giving up their baggage allowance to transport critical shipping documents. By delivering the original bill of lading by plane before the containers arrived by ship, DHL's innovative service sped up the customs clearance process and paved the way for the express delivery industry.
Even today, freight forwarders do not ship goods themselves. Instead, they facilitate the movement of goods by navigating the complex web of logistics networks for their customers. However, the freight forwarding business is still rooted much more in a traditional rather than a sharing model, even though it is asset light.
As we've seen in the hospitality, transportation, retail and entertainment industries, technology and social trends will likely provide new opportunities for the sharing economy in logistics. The mobile web and shifting values are changing the way we consume. Smartphones are ubiquitous and mobile technology is faster and more powerful than ever before. Convenience and instant gratification are in high demand and environmental awareness is at unprecedented levels. And as Millennials come of age, the world's first digital natives will increase demand and accelerate these trends. Sharing rather than owning will become the new normal.
The tremendous power of digital sharing platforms and crowd-based access to existing assets is rewriting the rules of business. New companies and business models are proliferating at a dizzying pace. The hospitality and transportation industries have been turned on their heads, with staffing, heavy industry, and – yes – logistics not far behind.
We're already seeing change creep slowly into the logistics industry. A number of current and potential use cases for sharing in logistics provide interesting insight into a future where access takes priority over ownership.
A truly shared warehouse, for example, reduces unused space, increases productivity, and cuts costs. Multi-customer warehouses are already commonplace, where third party logistics providers achieve greater economies of scale by consolidating demand, fulfillment, and logistics expertise at a single location. But space allocation is often inflexible and inefficient, as customers purchase a set amount that is not always fully utilized. Digital sharing platforms and next-generation inventory management tools can now provide unprecedented visibility, allowing warehouse managers to create a more dynamic business that opens a marketplace for excess warehouse space. DHL Supply Chain has pioneered a platform called DHL Spaces to do just that.
The rise of digital freight brokerage platforms is increasing interest in transport capacity sharing and reinventing the road freight industry as we know it – an industry plagued with inefficiency. For example, one in four trucks on the road in the United States and the European Union is empty. And those hauling cargo are on average only loaded to just over 50% capacity. Add the idle time resulting from traffic, loading, paper-based communications and documentation, and more, and you see an industry ripe for technology improvements and sharing. A number of digital freight platforms are now on the market, including DHL's own saloodo! In Europe, Freightos, Convoy, and Loadsmart in the United States. Following the sharing economy approach, these services leverage mobile technology to offer real-time data and communication between shippers and carriers to match loads with available capacity.
These are just two of the many examples of the sharing economy in logistics outlined in our new Trend Report: The Sharing Economy in Logistics – Rethinking logistics with access over ownership. The report, which aims to help you navigate through this new world, also includes best practices from other industries, such as hospitality, staffing, heavy industry, and transportation.
The landscape is changing rapidly and its imperative to understand the powerful forces driving these new business models and value innovation. We can learn a lot of lessons from those sectors that have already embraced sharing, not to mention the logistics industry innovators who are already applying the economic principles of sharing.
Technology and social trends are opening up a wealth of new opportunities to create and capture new value in future supply chains. The time has come to rethink our industry in the context of the sharing economy – to consider access over ownership.
Enjoyed your article! We have put capacity sharing into practice at FreshSpoke. We're making local food fresher and more accessible by providing wholesale buyers with the ability to source directly from local food producers and delivering to the door using the excess capacity that already exists in commercial delivery and warehousing sectors.
The listed concept of "sharing of resources" among the logistic providers is just great, the LP as of now are only sceptical about data security, once the same is secured you would see more and more Co's coming together bringing in economies of scale.