Meera Nanjundan

Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells

Arsenic trioxide (As2O3), used to treat promyelocytic leukemia, triggers cell death through unknown mechanisms. To further our understanding of As2O3-induced death, we analyzed its effects on transforming growth factor-β (TGFβ) signaling mediators in ovarian cells. Dysregulated TGFβ signaling is a characteristic of ovarian cancers. As2O3 reduced the protein expression of EVI1, TAK1, SMAD2/3, and TGFβRII while increasing SnoN/SkiL. EVI1 protein was modulated by treatment with the proteasome inhibitors, MG132 and PS-341/Velcade, suggesting that degradation occurs through the ubiquitin-proteasome pathway. The sensitivity of ovarian cells to As2O3-induced apoptosis correlated with expression of multidrug resistance protein 1. Interestingly, expression of SnoN was similar to LC3-II (autophagy marker), which increased with induction of cytoplasmic vacuolation preceding apoptosis. These vesicles were identified as autophagosomes based on transmission electron microscopy and immunofluorescence staining with EGFP–LC3. The addition of N-acetyl-L-cysteine (ROS scavenger) to As2O3-treated cells reversed changes in SnoN protein and the autophagic/apoptotic response. In contrast to beclin-1 knockdown, siRNA targeting ATG5, ATG7, and hVps34 markedly reduced autophagy in As2O3-treated ovarian carcinoma cells. Further, treatment with SnoN siRNA markedly decreased LC3-II levels and increased PARP degradation (an apoptosis marker). Collectively, these findings suggest that As2O3 induces a beclin-1-independent autophagic pathway in ovarian carcinoma cells and implicates SnoN in promoting As2O3-mediated autophagic cell survival.

Reciprocal Regulation of c-Src and STAT3 in Non-Small Cell Lung Cancer

Purpose: Signal transducer and activator of transcription-3 (STAT3) is downstream of growth factor and cytokine receptors, and regulates key oncogenic pathways in nonsmall cell lung cancer (NSCLC). Activation of STAT3 by cellular Src (c-Src) promotes tumor progression. We hypothesized that c-Src inhibition could activate STAT3 by inducing a homeostatic feedback loop, contributing to c-Src inhibitor resistance. Experimental Design: The effects of c-Src inhibition on total and phosphorylated STAT3 were measured in NSCLC cell lines and in murine xenograft models by Western blotting. c-Src and STAT3 activity as indicated by phosphorylation was determined in 46 human tumors and paired normal lung by reverse phase protein array. Modulation of dasatinib (c-Src inhibitor) cytotoxicity by STAT3 knockdown was measured by MTT, cell cycle, and apoptosis assays. Results: Depletion of c-Src by small interfering RNA or sustained inhibition by dasatinib increased pSTAT3, which could be blocked by inhibition of JAK. Similarly, in vivo pSTAT3 levels initially decreased but were strongly induced after sustained dasatinib treatment. In human tumors, phosphorylation of the autoinhibitory site of c-Src (Y527) correlated with STAT3 phosphorylation (r = 0.64; P = 2.5 106). STAT3 knockdown enhanced the cytotoxicity of dasatinib. Conclusions: c-Src inhibition leads to JAK-dependent STAT3 activation in vitro and in vivo. STAT3 knockdown enhances the cytotoxicity of dasatinib, suggesting a compensatory pathway that allows NSCLC survival. Data from human tumors showed a reciprocal regulation of c-Src and STAT3 activation, suggesting that this compensatory pathway functions in human NSCLC. These results provide a rationale for combining c-Src and STAT3 inhibition to improve clinical responses. (Clin Cancer Res 2009;15(22):685261)

Plasma Membrane Phospholipid Scramblase 1 Promotes EGF-dependent Activation of c-Src through the Epidermal Growth Factor Receptor

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1–/– embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.

Steroid receptor coactivator-3 expression in lung cancer and its role in the regulation of cancer cell survival and proliferation.

Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non-small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA-mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer.

Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms.

Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics.

Proteomic profiling identifies pathways dysregulated in non-small cell lung cancer and an inverse association of AMPK and adhesion pathways with recurrence.

Introduction: The identification of key pathways dysregulated in non-small cell lung cancer (NSCLC) is an important step toward understanding lung pathogenesis and developing new therapeutic approaches. Methods: Toward this goal, reverse-phase protein lysate arrays (RPPA) were used to compare signaling pathways between NSCLC tumors and paired normal lung tissue from 46 patients and assess their association with clinical outcome. Results: After RPPA quantification of 63 proteins and phosphoproteins, tissue pairs were randomized to a training set (n = 25 pairs) and test set (n = 21 pairs). In the training set, 15 protein markers were differentially expressed between tumors and normal lung (p ≤ 0.01), including markers in the PI3K/AKT and p38 MAPK signaling pathways (e.g., p70S6K, S6, p38, and phospho-p38), as well as caveolin-1 and &bgr;-catenin. A four-protein signature (p70S6K, cyclin B1, pSrc(Y527), and caveolin-1) independent of histology classified specimens as tumor versus normal with a predicted accuracy of 83%, sensitivity of 67%, and specificity of 100%. The signature was validated in the test set, correctly classifying all normal tissues and 14 of 21 tumor tissues. RPPA results were confirmed by immunohistochemistry for caveolin-1 and p70S6K. In tumors from patients with resected NSCLC, expression of proteins in the energy-sensing AMPK pathway (pLKB1, AMPK, p-Acetyl-CoA, pTSC2), adhesion, EGFR, and Rb signaling pathways was inversely associated with NSCLC recurrence. Conclusions: These data provide evidence for dysregulation of several pathways including those involving energy sensing and adhesion that are potentially associated with NSCLC pathogenesis and disease recurrence.

Roles and regulation of phospholipid scramblases

Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.

Overexpression of SnoN/SkiL, amplified at the 3q26.2 locus, in ovarian cancers: A role in ovarian pathogenesis

High‐resolution array comparative genomic hybridization of 235 serous epithelial ovarian cancers demonstrated a regional increase at 3q26.2 encompassing SnoN/SkiL, a coregulator of SMAD/TGFβ signaling. SnoN RNA transcripts were elevated in ∼80% of advanced stage serous epithelial ovarian cancers. In both immortalized normal (TIOSE) and ovarian carcinoma cell lines (OVCA), SnoN RNA levels were increased by TGFβ stimulation and altered by LY294002 and JNK II inhibitor treatment suggesting that the PI3K and JNK signaling pathways may regulate TGFβ‐induced increases in SnoN RNA. In TIOSE, SnoN protein levels were reduced 15min post TGFβ‐stimulation, likely by proteosome‐mediated degradation. In contrast, in OVCA, SnoN levels were elevated 3h post‐stimulation potentially as a result of inhibition of the proteosome. To elucidate the role of SnoN in ovarian tumorigenesis, we explored the effects of both increasing and decreasing SnoN levels. In both TIOSE and OVCA, SnoN siRNA decreased cell growth between 20 and 50% concurrent with increased p21 levels. In TIOSE, transient expression of SnoN repressed TGFβ induction of PAI‐1 promoters with little effect on the p21 promoter or resultant cell growth. In contrast to the effects of transient expression, stable expression of SnoN in TIOSE led to growth arrest through induction of senescence. Collectively, these results implicate SnoN levels in multiple roles during ovarian carcinogenesis: promoting cellular proliferation in ovarian cancer cells and as a positive mediator of cell cycle arrest and senescence in non‐transformed ovarian epithelial cells.

Iron Modulates Cell Survival in a Ras- and MAPK-Dependent Manner in Ovarian Cells

Ovarian cancer is a leading cause of cancer death in women in the United States. While the majority of ovarian cancers are serous, some rarer subtypes (i.e. clear cell) are often associated with endometriosis, a benign gynecological disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian cancers and induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells.

EVI1 splice variants modulate functional responses in ovarian cancer cells

Amplification of 3q26.2, found in many cancer lineages, is a frequent and early event in ovarian cancer. We previously defined the most frequent region of copy number increase at 3q26.2 to EVI1 (ecotropic viral integration site‐1) and MDS1 (myelodysplastic syndrome 1) (aka MECOM), an observation recently confirmed by the cancer genome atlas (TCGA). MECOM is increased at the DNA, RNA, and protein level and likely contributes to patient outcome. Herein, we report that EVI1 is aberrantly spliced, generating multiple variants including a Del190–515 variant (equivalent to previously reported) expressed in >90% of advanced stage serous epithelial ovarian cancers. Although EVI1Del190–515 lacks ∼70% of exon 7, it binds CtBP1 as well as SMAD3, important mediators of TGFβ signaling, similar to wild type EVI1. This contrasts with EVI1 1–268 which failed to interact with CtBP1. Interestingly, the EVI1Del190–515 splice variant preferentially localizes to PML nuclear bodies compared to wild type and EVI1Del427–515. While wild type EVI1 efficiently repressed TGFβ‐mediated AP‐1 (activator protein‐1) and plasminogen activator inhibitor‐1 (PAI‐1) promoters, EVI1Del190–515 elicited a slight increase in both promoter activities. Expression of EVI1 and EVI1Del427–515 (but not EVI1Del190–515) in OVCAR8 ovarian cancer cells increased cyclin E1 LMW expression and cell cycle progression. Furthermore, knockdown of specific EVI1 splice variants (both MDS1/EVI1 and EVI1Del190–515) markedly increased claudin‐1 mRNA and protein expression in HEY ovarian and MDA‐MB‐231 breast cancer cells. Changes in claudin‐1 were associated with alterations in specific epithelial–mesenchymal transition markers concurrent with reduced migratory potential. Collectively, EVI1 is frequently aberrantly spliced in ovarian cancer with specific forms eliciting altered functions which could potentially contribute to ovarian cancer pathophysiology.