TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2.

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target

(a) Caspase-3 cleavage after Staurosporine treatment: The left panel shows Caspase-3 cleavage in H2009 and its clones treated with 0.5μM Staurosporine for 3 hours. The right panel shows Caspase-3 cleavage after ABT-737 and Staurosporine treatment, where more caspase cleavage is observed in HB6 and HB1. Graph represents densitometric analysis of the cleaved caspase western blot in each panel. Data represents independent triplicate determinations ± SEM (standard error of mean). (b) TIMP-1 inhibits apoptosis induced by Staurosporine: TIMP-1 overexpression does not result in significant PARP-1 cleavage after 3 hrs of 0.5μM Staurosporine treatment, probably due to upregulation of Bcl-2, which plays an inhibitory role. Expression of Bcl-xL was unchanged. The side panel shows the graphical representation of cleaved PARP expression (p<0.0001, ANOVA). Data is representative of three independent experiments. C = control, S = Staurosporine treated. (c) Abrogating Bcl-2 by ABT-737 restores the PAR

<p>(a) Caspase-3 cleavage after Staurosporine treatment: The left panel shows Caspase-3 cleavage in H2009 and its clones treated with 0.5μM Staurosporine for 3 hours. The right panel shows Caspase-3 cleavage after ABT-737 and Staurosporine treatment, where more caspase cleavage is observed in HB6 and HB1. Graph represents densitometric analysis of the cleaved caspase western blot in each panel. Data represents independent triplicate determinations ± SEM (standard error of mean). (b) TIMP-1 inhibits apoptosis induced by Staurosporine: TIMP-1 overexpression does not result in significant PARP-1 cleavage after 3 hrs of 0.5μM Staurosporine treatment, probably due to upregulation of Bcl-2, which plays an inhibitory role. Expression of Bcl-xL was unchanged. The side panel shows the graphical representation of cleaved PARP expression (p<0.0001, ANOVA). Data is representative of three independent experiments. C = control, S = Staurosporine treated. (c) Abrogating Bcl-2 by ABT-737 restores the PARP cleavage pattern: Treatment with 1μM ABT-737 for 24 hrs followed by 0.5μM Staurosporine from 21^st hour increased PARP cleavage in HB1 and HB6 cells. (d) Antiapoptotic function of TIMP-1 is independent of MMP activity: The T2G mutated TIMP-1 overexpressing clone showed similar pattern of PARP cleavage indicating that this activity of TIMP-1 was MMP independent.</p

(a) Comparison of exogenous (in SFCM) and endogenous (cell lysates) TIMP-1 in H2009, empty vector and TIMP-1 overexpressing clones: Cells were grown overnight in serum free media to assess exogenous TIMP-1 and cell lysates were collected from cells grown in complete media. The conditioned media and the cell lysates from the cell lines were assayed for TIMP-1 by ELISA. The X-axis shows H2009 cell line and its clones. The Y-axis shows concentration of TIMP-1 in ng/mL. A significant upregulation of TIMP-1 in overexpressed clones is seen (p<0.01, Student’s t test). Data represents independent triplicate determinations ± SEM (standard error of mean). (b) Effect of ABT-737 and Staurosporine on apoptosis: Cells were first treated with ABT-737 (1 μM, for 21 hours) following which Staurosporine was added at 0.5μM concentration for 3 hours (all apoptosis related experiments follow this regimen unless mentioned otherwise). Cell apoptosis was evaluated by Hoechst 33258 staining. The control cells

<p>(a) Comparison of exogenous (in SFCM) and endogenous (cell lysates) TIMP-1 in H2009, empty vector and TIMP-1 overexpressing clones: Cells were grown overnight in serum free media to assess exogenous TIMP-1 and cell lysates were collected from cells grown in complete media. The conditioned media and the cell lysates from the cell lines were assayed for TIMP-1 by ELISA. The X-axis shows H2009 cell line and its clones. The Y-axis shows concentration of TIMP-1 in ng/mL. A significant upregulation of TIMP-1 in overexpressed clones is seen (p<0.01, Student’s t test). Data represents independent triplicate determinations ± SEM (standard error of mean). (b) Effect of ABT-737 and Staurosporine on apoptosis: Cells were first treated with ABT-737 (1 μM, for 21 hours) following which Staurosporine was added at 0.5μM concentration for 3 hours (all apoptosis related experiments follow this regimen unless mentioned otherwise). Cell apoptosis was evaluated by Hoechst 33258 staining. The control cells displayed normal nuclei. Staurosporine treatment with or without ABT-737 showed significantly more condensed and bright fluorescent nuclei with nuclear fragmentation in H2009 and HEV cells. The number of apoptotic cells were less in the HB1 and HB6 cells in panel C and D as shown by the arrows. (c) Quantitative representation of Hoechst 33258 apoptosis assay: Hoechst staining showed significantly more apoptosis in H2009 cells and the empty vector clones (HEV), compared to the TIMP-1 overexpressing clones(HB1, HB6), by One Way ANOVA (p<0.05), with or without staurosporine treatment. The control groups (H2009, HEV) showed almost 2-fold greater apoptotic morphology compared to TIMP-1 overexpressing clones. Data is representative of 3 independent experiments ± SEM. (d) TIMP-1 overexpressing cell lines show increased expression of Bcl-2 mRNA in response to staurosporine treatment: H2009 cells and its clones were treated with 0.5μM Staurosporine (S) and subjected to apoptosis specific PCR array. A significant 3 fold increase in Bcl-2 and TIMP1, was observed and confirmed by qPCR (p<0.01, One Way ANOVA with posthoc Dunnett’s test). (e) Expression of Bcl-2 and TIMP-1 in H2009 cells: The top panel shows semiquantitative reverse transcription PCR after 3hrs of Staurosporine (S) treatment. An increased amount of Bcl-2 is observed in the TIMP-1 overexpressing clones. Lower panel indicates an increased amount of Bcl-2 in the TIMP-1 overexpressing clones by western blot. The TIMP-2 level remained unchanged. The relative protein band density was normalized to β-Actin. Data is representative of triplicate independent experiments.</p

Antiapoptotic function of TIMP-1 does not involve the Akt pathway.

<p>TIMP-1 overexpression activates p90RSK which in turn activates BAD, concomitantly reducing Bax expression in the H2009 cells. The right panel shows treatment with ABT-737 and Staurosporine (AS) restores the p-p90RSK, p-BAD and Bax levels. Fig 4 also shows absence of Akt involvement.</p

Somatic Mutations, Allele Loss, and DNA Methylation of the Cub and Sushi Multiple Domains 1 (CSMD1) Gene Reveals Association with Early Age of Diagnosis in Colorectal Cancer Patients

<div><p>Background</p><p>The Cub and Sushi Multiple Domains 1 (CSMD1) gene, located on the short arm of chromosome 8, codes for a type I transmembrane protein whose function is currently unknown. CSMD1 expression is frequently lost in many epithelial cancers. Our goal was to characterize the relationships between CSMD1 somatic mutations, allele imbalance, DNA methylation, and the clinical characteristics in colorectal cancer patients.</p> <p>Methods</p><p>We sequenced the CSMD1 coding regions in 54 colorectal tumors using the 454FLX pyrosequencing platform to interrogate 72 amplicons covering the entire coding sequence. We used heterozygous SNP allele ratios at multiple CSMD1 loci to determine allelic balance and infer loss of heterozygosity. Finally, we performed methylation-specific PCR on 76 colorectal tumors to determine DNA methylation status for CSMD1 and known methylation targets ALX4, RUNX3, NEUROG1, and CDKN2A.</p> <p>Results</p><p>Using 454FLX sequencing and confirming with Sanger sequencing, 16 CSMD1 somatic mutations were identified in 6 of the 54 colorectal tumors (11%). The nonsynonymous to synonymous mutation ratio of the 16 somatic mutations was 15∶1, a ratio significantly higher than the expected 2∶1 ratio (p = 0.014). This ratio indicates a presence of positive selection for mutations in the CSMD1 protein sequence. CSMD1 allelic imbalance was present in 19 of 37 informative cases (56%). Patients with allelic imbalance and CSMD1 mutations were significantly younger (average age, 41 years) than those without somatic mutations (average age, 68 years). The majority of tumors were methylated at one or more CpG loci within the CSMD1 coding sequence, and CSMD1 methylation significantly correlated with two known methylation targets ALX4 and RUNX3. C:G>T:A substitutions were significantly overrepresented (47%), suggesting extensive cytosine methylation predisposing to somatic mutations.</p> <p>Conclusions</p><p>Deep amplicon sequencing and methylation-specific PCR reveal that CSMD1 alterations can correlate with earlier clinical presentation in colorectal tumors, thus further implicating CSMD1 as a tumor suppressor gene.</p> </div

(a): Interdependency of TIMP-1 and Bcl-2 proteins: H2009 cells and its clones were treated with 0.5μM Staurosporine (S), which showed a reduction of TIMP-1 protein expression. Treatment with a BH3 mimetic ABT-737(A) reduced TIMP-1 protein levels compared to the controls. Combined treatment with ABT-737 and Staurosporine (AS) showed a marked reduction of TIMP-1 expression, without any changes in TIMP-2. (b) TIMP-1 mRNA expression level after treatment with Staurosporine and ABT-737: HB1 and HB6 TIMP-1 levels were reduced with combined ABT-737 and Staurosporine treatment. Two Way Repeated Measure ANOVA showed p = 0.0063, indicating a very significant down regulation of relative expression of TIMP1 in the overexpressed clones after ABT+Staurosporine treatment indicating down-regulating Bcl2 affects TIMP1 level. (c) Co-immunoprecipitation of TIMP-1 and Bcl-2: A possible interaction between the Bcl-2 and TIMP-1 revealed by co-immunoprecipitation. Cell extracts from H2009 and it’s clones wer

<p>(a): Interdependency of TIMP-1 and Bcl-2 proteins: H2009 cells and its clones were treated with 0.5μM Staurosporine (S), which showed a reduction of TIMP-1 protein expression. Treatment with a BH3 mimetic ABT-737(A) reduced TIMP-1 protein levels compared to the controls. Combined treatment with ABT-737 and Staurosporine (AS) showed a marked reduction of TIMP-1 expression, without any changes in TIMP-2. (b) TIMP-1 mRNA expression level after treatment with Staurosporine and ABT-737: HB1 and HB6 TIMP-1 levels were reduced with combined ABT-737 and Staurosporine treatment. Two Way Repeated Measure ANOVA showed p = 0.0063, indicating a very significant down regulation of relative expression of TIMP1 in the overexpressed clones after ABT+Staurosporine treatment indicating down-regulating Bcl2 affects TIMP1 level. (c) Co-immunoprecipitation of TIMP-1 and Bcl-2: A possible interaction between the Bcl-2 and TIMP-1 revealed by co-immunoprecipitation. Cell extracts from H2009 and it’s clones were immunoprecipitated (IP) with anti-Bcl-2 or normal rabbit IgG as a negative control. The immunoblot (IB) was probed for TIMP-1, TIMP-2 and Bcl-2. No TIMP-2 was pulled down with Bcl-2, showing that only TIMP-1 is associated with Bcl-2 when apoptosis is induced. Input represents 60μg of cell extract.</p

Sequential Probability Ratio Test (SPRT) analysis of CSMD1 based on digital allele counting.

<p>The upper bound curve shown in the graph demonstrates the 95% CI threshold of CSMD1 loci being classified as unbalanced, whereas the lower bound curve demonstrates the 95% CI threshold of CSMD1 loci being classified as balanced. Tumors with two or more contiguous loci that were imbalanced were classified as having undergone loss of heterozygosity. Loci that fell between the two thresholds were deemed indeterminate for allelic characterization. Certain loci in CSMD1 demonstrated allelic balance, though they resided in tumors that were unbalanced for CSMD1. This result alludes to chromosomal breaks that occur within the CSMD1 gene sequence, downstream of the examined loci.</p

CSMD1 mutation frequencies of somatic variants in Tumor 517.

<p>Ten CSMD1 somatic mutation were found in Tumor 517. However, these particular mutations occur at varying subsets of concentrations, indicating that each subset may have arisen from an independent clone within the tumor population.</p