- Research article
- Open Access
- Open Peer Review
Targeting cell migration and the endoplasmic reticulum stress response with calmodulin antagonists: a clinically tested small molecule phenocopy of SEC62 gene silencing in human tumor cells
- Maximilian Linxweiler†1,
- Stefan Schorr†1,
- Nico Schäuble1,
- Martin Jung1,
- Johannes Linxweiler1,
- Frank Langer2,
- Hans-Joachim Schäfers2,
- Adolfo Cavalié3,
- Richard Zimmermann1 and
- Markus Greiner1Email author
© Linxweiler et al.; licensee BioMed Central Ltd. 2013
- Received: 10 April 2013
- Accepted: 27 November 2013
- Published: 5 December 2013
Tumor cells benefit from their ability to avoid apoptosis and invade other tissues. The endoplasmic reticulum (ER) membrane protein Sec62 is a key player in these processes. Sec62 is essential for cell migration and protects tumor cells against thapsigargin-induced ER stress, which are both linked to cytosolic Ca2+. SEC62 silencing leads to elevated cytosolic Ca2+ and increased ER Ca2+ leakage after thapsigargin treatment. Sec62 protein levels are significantly increased in different tumors, including prostate, lung and thyroid cancer.
In lung cancer, the influence of Sec62 protein levels on patient survival was analyzed using the Kaplan-Meier method and log-rank test. To elucidate the underlying pathophysiological functions of Sec62, Ca2+ imaging techniques, real-time cell analysis and cell migration assays were performed. The effects of treatment with the calmodulin antagonists, trifluoperazine (TFP) and ophiobolin A, on cellular Ca2+ homeostasis, cell growth and cell migration were compared with the effects of siRNA-mediated Sec62 depletion or the expression of a mutated SEC62 variant in vitro. Using Biacore analysis we examined the Ca2+-sensitive interaction of Sec62 with the Sec61 complex.
Sec62 overproduction significantly correlated with reduced patient survival. Therefore, Sec62 is not only a predictive marker for this type of tumor, but also an interesting therapeutic target. The present study suggests a regulatory function for Sec62 in the major Ca2+ leakage channel in the ER, Sec61, by a direct and Ca2+-sensitive interaction. A Ca2+-binding motif in Sec62 is essential for its molecular function. Treatment of cells with calmodulin antagonists mimicked Sec62 depletion by inhibiting cell migration and rendering the cells sensitive to thapsigargin treatment.
Targeting tumors that overproduce Sec62 with calmodulin antagonists in combination with targeted thapsigargin analogues may offer novel personalized therapeutic options.
- Endoplasmic reticulum (ER) stress
- Cell migration
- Ca2+ homeostasis
- Calmodulin antagonists
Cancer is one of the most common deadly diseases , and the proportion of patients dying because of malignant disease is increasing every year . Lung cancer is of particular concern with a five-year survival rate below 20% . Therapeutic opportunities are scarce for patients suffering from squamous cell carcinoma (SCC) of the lung . We have recently reported SEC62 as a new candidate oncogene, as it is significantly overexpressed with elevated protein levels in SCC .
Sec62 is an essential protein in yeast and part of the Sec62/Sec63 sub-complex of the SEC complex, acting as a docking site for posttranslational protein transport . Studies in mammals have shown that Sec62 is associated with the heterotrimeric Sec61 complex and Sec63 [7, 8], and that it participates in the targeting and translocation of small pre-secretory proteins to the endoplasmic reticulum (ER) [9, 10]. Mammalian Sec62 can also interact with the ribosome, thereby regulating translation . Elevated Sec62 protein levels are functionally linked to increased cell migration capability  and reduced sensitivity to thapsigargin-induced ER stress , both of which are tightly regulated by the cytosolic Ca2+ concentration [14–16]. Previously, we have shown that reduced Sec62 protein levels lead to an at least two-fold increase in basal cytosolic Ca2+ and a much greater increase in cytosolic Ca2+ concentration in response to thapsigargin treatment (i.e., increased ER Ca2+ leakage) . These results demonstrate a significant influence of Sec62 on ER Ca2+ homeostasis, making Sec62 a promising target for new therapeutic approaches. Regulation of cytosolic Ca2+ levels by targeting this protein may induce anti-metastatic and anti-proliferative effects.
In the present study, we used small molecule inhibitors of the Ca2+-binding protein, calmodulin, to mimic the phenotypes previously observed after SEC62 silencing. This approach provided new insight into the physiological function of Sec62 and may lead to a new therapeutic strategy for personalized cancer therapy.
Cell culture and tissue samples
PC3 (DSMZ no. ACC 465), HeLa (DSMZ no. ACC 57), A549 (DSMZ no. ACC 107), BC01 (kindly provided by G. Unteregger, Saarland University Hospital, Department of Urology and Pediactric Urology), BHT 101 (DSMZ no. ACC 279), ML1 (DSMZ no. ACC 464) and HEK293 (DSMZ no. ACC 305) cells were cultured at 37°C in DMEM medium (Gibco Invitrogen, Karlsruhe, Germany) containing 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (PAA, Pasching, Austria) in a humidified environment with 5% CO2. H1299 cells (ATCC no. CRL-5803D) were cultured in RPMI1640 medium (PAA) containing the same supplements. We used stably transfected HEK293 cells expressing plasmid-encoded wild-type SEC62 (pSEC62-IRES-GPF) or an empty control plasmid (pIRES-GPF) . A plasmid encoding SEC62 with a D308A point mutation (pSEC62 D308A -IRES-GPF) was generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The plasmid was sequenced to confirm the point mutation. A stably transfected cell line expressing this mutant gene was generated by transfecting 2.4 × 105 HEK293 cells in a 6-well plate using FuGeneHD Reagent (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. After 72 h, the medium was replaced with normal culture medium containing 1% G418 and the cells were cultured until selection was achieved. After harvesting, the cells were diluted to a density of 1 cell per 100 μl, and 100 μl were seeded in each well of a 96-well plate in medium containing 1% G418. Clones originating from a single cell were selected and analyzed for Sec62 content. All experiments using stably transfected cell lines were performed in normal growth medium containing 1% G418. Stably transfected HEK293 cells were used for migration assays, as transient transfection or treatment with FuGeneHD transfection reagent strongly inhibits cell migration.
We analyzed Sec62 levels in cancerous and tumor-free lung tissue from 70 non-small cell lung cancer (NSCLC) patients with pathologically confirmed adenocarcinoma (AC) or squamous cell carcinoma (SCC) using western blot with β-actin as a loading control. We calculated the relative elevation in the Sec62 protein content (rSec62 = [Sec62tumor/b-actintumor]/[Sec62tumor-free/b-actintumor-free]) in the tumor . All patients (n = 70) and the subgroups of AC (n = 35) and SCC (n = 35) patients were divided into two groups based on the median rSec62 value, and survival analyses were performed using the Kaplan-Meier method and the log-rank test. Only samples from patients who gave signed informed consent were used. All samples were received for therapeutic or diagnostic purpose and anonymized. Therefore, according to the guidelines of the local ethics board (“Ethikkommision der Ärztekammer des Saarlandes”) and the statement of the national ethics committee (nationaler Ethikrat (Hrsg.): Biobanken für die Forschung. Stellungnahme. Berlin 2004 [http://www.ethikrat.org/dateien/pdf/NER_Stellungnahme_Biobanken.pdf]) they can be used without specific approval by an ethics board.
Protein in lysates from 2 × 105 cultured cells was quantified by western blot analysis. We used an affinity-purified polyclonal rabbit anti-peptide antibody directed against the C-terminus of human Sec62, a polyclonal rabbit anti-BiP antibody, a polyclonal rabbit anti-peptide antibody directed against the C-terminus of human Sec61α, and a monoclonal murine anti-β-actin antibody (Sigma Aldrich, Taufkirchen, Germany, A5441-.5ML). The primary antibodies were visualized using an ECLTM Plex goat anti-rabbit IgG-Cy5 or ECLTM Plex goat anti-mouse IgG-Cy3 conjugate (GE Healthcare, Munich, Germany), and the Typhoon-Trio imaging system (GE Healthcare) in combination with Image Quant TL software 7.0 (GE Healthcare). We determined the ratio of Sec62, Sec61α and BiP relative to β-actin.
Silencing of gene expression by siRNA
For gene silencing, 5.4 × 105 cells were seeded in 6-cm dishes containing normal culture medium. The cells were transfected with SEC62-UTR siRNA (CGUAAAGUGUAUUCUGUACtt; Ambion, Life Technologies, Carlsbad, CA, USA), SEC62 siRNA (GGCUGUGGCCAAGUAUCUUtt; Ambion), SEC61A1 siRNA (GGAAUUUGCCUGCUAAUCAtt, QIAGEN, Hilden, Germany), or control siRNA (AllStars Neg. Control siRNA; QIAGEN) using HiPerFect Reagent (QIAGEN) according to the manufacturer’s instructions. After 24 h, the medium was changed and the cells were transfected a second time. Silencing efficiency was evaluated by western blot analysis. The maximum silencing effect was seen 72 h (SEC62 siRNAs) or 96 h (SEC61A1 siRNA) after the first transfection.
Real-time cell proliferation analysis
The xCELLigence SP system (Roche Diagnostics GmbH, Mannheim, Germany) was used for real-time analysis of cell proliferation. In this system, 1.0 × 104 or 2.0 × 104 stably transfected HEK293 cells, untreated HEK293, PC3 or HeLa cells, or PC3 cells pretreated with siRNA in 6-cm dishes were seeded into a 96-well e-plate (Roche Diagnostics GmbH) according to the manufacturer’s instructions. Cells pretreated with siRNA were seeded 24 h after the second transfection. When cells were treated with thapsigargin, TFP or ophiobolin A, the treatment was performed at least 4 h after seeding the plates. Cell proliferation was monitored for 53–96 h and the data was evaluated with RTCA 1.2 software (Roche Diagnostics GmbH). Thapsigargin was used at concentrations of 6 or 10 nM, because these concentrations did not affect cell growth. This is in contrast to the live-cell calcium imaging experiments, where 1 μM thapsigargin was used to visualize short-term calcium effects monitored only over a time span of up to 1200 s.
Peptide spot binding assay
Thirteen peptides spanning the N-terminus of the human Sec61α protein were synthesized on cellulose membranes via a C-terminal attachment as described previously [17, 18]. The peptides consisted of 12 amino acid residues with an overlap of 10 residues and were incubated in binding buffer (30 mM Tris–HCl, pH 7.4, 170 mM NaCl, 6.4 mM KCl, 5% sucrose, 0.05% Tween20) with Sec62-C-6His (1 μM), which was purified from Escherichia coli as described previously . To detect bound protein, the membranes were washed twice with binding buffer, incubated with anti-His-POD-coupled antibody (1:1000, QIAGEN), washed twice with binding buffer again, incubated with ECL (GE Healthcare) and visualized using a lumi-imaging system (Roche Diagnostics GmbH).
Surface plasmon resonance spectroscopy
Surface plasmon resonance (SPR) spectroscopy was performed in a BIAlite upgrade system (Biacore, Freiburg, Gerrmany). Peptides representing the N-terminus of Sec61 (AIKFLEVIKPFC) or the N-terminus of TRAM (VLSHEFELQNGADC) were immobilized in the measuring cell or control cell, respectively, on a CM5 sensor chip using ligand-thiol-coupling according to the manufacturer’s protocol. Measurements were performed at a flow rate of 10 μl/min in a Ca2+−free buffer containing 10 mM HEPES-KOH, pH 7.4, 150 mM NaCl, 2 mM MgCl, 6.4 mM KCl and 0.005% surfactant. For interaction analysis, E. coli-purified Sec62-C-6His (1 μM)  in buffer minus Ca2+ or in the same buffer containing 2 mM Ca2+, or the Ca2+-containing buffer alone was passed over the chip. Response units are shown as the difference between the measuring and control cells. The analysis was carried out using BIA evaluation software version 3.1 (Biacore) with 1:1 binding models and mass transfer.
Migration potential analysis
Migration was tested using the BD Falcon FluoroBlok system (BD, Franklin Lakes, NJ, USA) in 24-well inserts. A total of 2.5 × 104 stably transfected HEK293 cells, or untreated PC3 or HeLa cells were loaded in normal medium containing 0.5% FBS. When DMSO, TFP or ophiobolin A was used, the drugs were added to the top and bottom chambers at various concentrations. The inserts were placed in medium with 10% FBS as a chemoattractant. After 72 h, the cells were fixed with methanol and stained with DAPI, and migrating cells were analyzed on the back of the membrane using fluorescence microscopy.
Live-cell calcium imaging
For live-cell Ca2+ imaging, HeLa cells were loaded with 4 μM FURA-2 AM (Molecular Probes, Eugene, OR, USA) in DMEM for 45 min at room temperature as described previously [19, 20]. Two washes were performed with a Ca2+-free buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM EGTA and 10 mM glucose in 10 mM HEPES-KOH, pH 7.35) and the experiments were carried out in the same solution. A ratiometric measurement was performed for 3 min to determine the initial cytosolic [Ca2+]. The measurement was continued after the addition of 1 μM thapsigargin or - to measure store operated calcium entry (SOCE) - 2.5 mM Ca2+. Cells pretreated as described in the text were compared with respect to the initial cytosolic [Ca2+] and thapsigargin-induced changes in cytosolic [Ca2+]. Data were collected by an iMIC microscope and polychromator V (Till Photonics, Graefelfing, Germany) by alternating excitation between 340 and 380 nm, and measuring the emitted fluorescence at 510 nm (dichroic, DCLP410; emitter filter LP470; Till Photonics). Images containing 50–60 cells/frame were sampled every 3 sec. FURA-2 signals were recorded as an F340/F380 ratio, where F340 and F380 correspond to the background-subtracted fluorescence intensities at 340 and 380 nm, respectively. The cytosolic [Ca2+] was estimated from the ratio measurements using an established calibration method .
ER luminal Ca2+ was determined using HeLa-CES2 cells that contain ER lumenal carboxylesterase and allow efficient dye loading of the ER, as previously described . Cells were loaded with 4 μM Fluo5N AM (solubilized in Pluronic F-127) in HBSS (Gibco) for 15 min at 37°C, washed with HBSS and incubated for another 30 min at 25°C to remove remaining cytosolic dye. After 1 min incubation in Ca2+-free buffer, buffer (0.1% DMSO, solvent control), ophiobolin A (100 μM) or TFP (10 μM) were added, samples were measured for 2 min, and then 1 μM thapsigargin was added to unmask the passive Ca2+ efflux from the ER. After 8 min, 5 μM ionomycin was applied to release the total ER Ca2+ of the cells. Data were collected by the iMIC microscope with excitation at 490 nm and measurement of the emitted fluorescence at 530 nm. Images containing 10–25 cells/frame were sampled every 3 s. A τ1/2-value was calculated for each curve as the time point at which 50% reduction of fluorescence signal was achieved after addition of thapsigargin.
Data were analyzed using Excel 2007 and Origin 6.1.
Sec62 levels in cancer tissue predicts survival of NSCLC patients
Treatment with calmodulin antagonists mimics changes in the cytosolic calcium concentration induced by SEC62 silencing
To verify that indeed Ca2+ leakage from the ER is responsible for the increase in cytosolic Ca2+ concentration after treatment with ophiobolin A or TFP, we first used HeLa-CES2 cells in combination with Fluo5N to directly measure changes in ER luminal Ca2+. We observed an initial Ca2+-release from the ER after addition of calmodulin antagonists and a significantly higher efflux in the ophiobolin A or TFP pretreated cells in response to thapsigargin (Figure 2B), with τ1/2-values of 163 s for the buffer control, 87 s after pretreatment with ophiobolin A and 65 s after pretreatment with TFP. Next, we asked if the calmodulin antagonists influence the store operated calcium entry (SOCE). To this end, we measured the cytosolic Ca2+ concentration after treating the cells externally with a Ca2+-containing buffer instead of thapsigargin and EGTA. These experiments disclosed that SOCE was also significantly stimulated by pretreatment with calmodulin antagonists. Moreover, a comparison between cells treated with control siRNA and cells treated with two different siRNAs directed against SEC61A1 indicated a crucial function of the Sec61 channel in SOCE under these conditions (Figure 2C). We note that we used a HeLa cell-based model system rather than lung cancer cells for two main reasons. First, the HeLa cells provide a well-established model system for SEC61A1 or SEC62 gene silencing, and live-cell Ca2+ imaging. Second, we were able to compare the results of live-cell Ca2+ imaging experiments on cells treated with SEC61A1 or SEC62 siRNA with our previous observations (Figure 2A–D) [13, 24].
Furthermore, we examined whether the effect of Sec62 on ER Ca2+ leakage can be linked to the Ca2+-permeable Sec61 complex as has been previously shown for the effects of TFP and ophiobolin A [17, 24]. To address this question, we treated HeLa cells for 96 h with SEC62 siRNA, SEC61A1 siRNA, SEC62 plus SEC61A1 siRNA, or a negative control siRNA. Simultaneous silencing of SEC61A1 and SEC62 by siRNA had an inhibitory effect on SEC62 silencing-induced Ca2+ efflux (Figure 2D). Western blot analysis indicated that the silencing efficiency of both siRNAs was > 80% (Figure 2D, insert). Thus, calmodulin antagonists and Sec62 contribute to reducing Ca2+ leakage from the ER at the Sec61 complex level. As has already been shown for calmodulin , Sec62 presumably acts by direct interaction with Sec61α.
Mutation in a predicted calcium-binding motif in the C-terminal domain of Sec62 leads to a dominant-negative effect on cell migration and ER calcium leakage
Previously, we showed that Sec62 depletion inhibits the spread of metastatic tumor cells and increases cell sensitivity to Ca2+-driven ER stress [12, 13]. By introducing the D308A mutation into the predicted Ca2+-binding motif within the C-terminal domain of Sec62, we confirmed the function of Sec62 in regulating ER Ca2+ homeostasis (Figure 3A). In this experiment, the expression of plasmid-encoded SEC62-WT or SEC62 D308A was evaluated by quantitative western blot analysis of the stably transfected HEK293 cell lines. We observed a nine-fold increase in Sec62 in the presence of pSEC62-WT and an almost five-fold increase in Sec62 in the presence of pSEC62 D308A in comparison with the control plasmid (Figure 3B). We then compared stably transfected HEK293 cells overexpressing the plasmid-encoded mutant SEC62 (pSEC62 D308A -IRES-GFP) with cells overexpressing SEC62-WT (pSEC62-IRES-GFP). Overproduction of Sec62-WT led to increased migration, which is in agreement with our previous observations . In contrast, overproduction of the mutant Sec62 protein, even in the presence of the endogenous Sec62-WT protein, reduced cell migration in a manner similar to SEC62 silencing (Figure 3C). Also, the sensitivity to thapsigargin (Figure 3D) and thapsigargin-induced Ca2+ leakage from the ER increased after SEC62 D308A expression (Figure 3E). Overall, SEC62-WT overexpression did not affect cell growth or ER Ca2+ leakage, whereas SEC62 D308A overexpression led to a phenotype comparable to that of SEC62 silencing. These experiments clearly indicate a direct influence of the predicted EF hand motif in Sec62 on ER Ca2+ homeostasis and its direct connection to the observed phenotypes.
HeLa and HEK293 cells are more sensitive to TFP treatment than PC3 cells
Treatment with calmodulin antagonists and SEC62 silencing result in comparable cellular phenotypes
Because Sec62 depletion by siRNA transfection alone was sufficient to block cell migration in previous experiments , we tested whether SEC62 overexpression can rescue ophiobolin A- or TFP-treated cells. We used HEK293 cells, which only poorly migrate without treatment but can be stimulated to migrate by the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA), a drug that down-regulates agonist-driven Ca2+ release from the ER  and stimulates cell migration [26, 27]. We compared HEK293 cells stably transfected with a pIRES-GFP vector (control plasmid) and HEK293 cells stably overexpressing plasmid-encoded SEC62 (pSEC62-IRES-GPF). The migration of the control plasmid-transfected HEK293 cells was completely inhibited by 100 nM ophiobolin A or 8 μM TFP (Figure 5B and C). However, cells overexpressing SEC62 still migrated under these conditions (Figure 5B and C), indicating that the Sec62 protein content resulted in higher cell resistance to treatment with calmodulin antagonists. Quantitative western blot analysis confirmed a four-fold increase in Sec62 in the pSEC62-WT-carrying HEK293 cells (Figure 5D). These observations support a Ca2+-dependent influence of Sec62 on cell migration.
Growth inhibition induced by calmodulin antagonists is enhanced by Sec62 depletion
Sec62 as a new prognostic marker for NSCLC patients
Because SEC62 silencing inhibits cancer cell migration and increases sensitivity to Ca2+-driven cellular stress, we investigated whether Sec62 represents not only a possible new target for anti-cancer therapies, but also a prognostic marker for lung cancer patients. A low rSec62 value predicts increased survival of NSCLC patients, with an even stronger predictive potential for SCC patients. Together with our previous findings that SEC62 is overexpressed and correlates with lymph node metastasis (N + vs. N0) and cancer progression (G3 vs. G2) in SCC of the lung , the results indicate that Sec62 plays a crucial role in lung cancer biology and is both a promising new target for cancer therapy and a reliable marker of clinical outcomes. Additional studies are needed to determine whether the role of Sec62 as a prognostic marker is solely because of the tumor cells’ dependency on a sufficient Sec62 level to enable metastasis and resistance to Ca2+-driven cellular stress, or whether Sec62 has additional contributing functions.
Phenotypic analogy of cellular calcium changes following treatment with calmodulin antagonists provides new insight into molecular events in Sec62-depleted cells
Furthermore, the dominant-negative phenotype induced by mutation of the predicted EF hand motif in the Sec62 protein, which was completely congruent with the effects of Sec62 depletion or treatment with calmodulin antagonists, strongly points to a direct regulation of Sec62 function by Ca2+ binding to the motif. The Sec61 complex has recently been shown to form an important Ca2+ leakage channel in the ER, the major cellular Ca2+ reservoir, and that Ca2+ efflux via this polypeptide pore is regulated by calmodulin  and the ER luminal Hsp70 chaperone, BiP . Taken together with our new findings that Ca2+ efflux from the ER after Sec62 depletion occurs through the Sec61 complex, we propose a model in which Sec62 is an additional regulator of the Sec61 Ca2+ leakage channel. Sec62 regulates Ca2+ leakage via a direct interaction with Sec61. The association of these two proteins has already been demonstrated [7, 8] and has been found to be Ca2+ sensitive (Figure 2D). Following our model, Sec62 senses emanating Ca2+ via a microdomain in close proximity to the Sec61 channel. After Ca2+ binding, Sec62 binding to Sec61 is relieved, thereby uncovering the binding site and facilitating the binding of Ca2+-calmodulin to Sec61 on the cytosolic surface of the ER, leading to closure of the channel (Figure 7A and E). In this model, the Sec62 variant with the mutated EF hand (Sec62D308A) is no longer able to sense the emanating Ca2+, and thus closure of the Sec61 channel by Ca2+-calmodulin binding would not occur, which explains the increased Ca2+ response observed in our live-cell Ca2+ imaging experiments. An additional mode of action of Sec62 on the luminal side is possible via a role in the recruitment of BiP as a Ca2+ efflux-limiting factor via its interaction with the J-domain-containing Hsp40 protein, Sec63 [7, 8, 11, 32].
Mimicking the Sec62-depletion phenotype with small molecule treatment as a possible new therapeutic option for cancer patients
Previous studies have shown that Sec62 depletion by transfection with SEC62 siRNA leads to cell migration inhibition and higher sensitivity to ER stress induced by Ca2+ dysregulation [5, 12, 13]. Therefore, SEC62 silencing seems to provide a potential approach for cancer treatment, especially lung and thyroid cancer, as such treatment could lead to reduced metastatic spread of tumor cells and higher sensitivity to chemotherapies working via the induction of ER stress. However, despite intensive studies over the past few decades [33–36], RNA interference remains unfeasible for clinical treatment of human diseases, mainly because of toxic side effects and problems in achieving adequate concentrations in the target tissues . Our present results provide a potential strategy for overcoming these problems with tumors that overproduce Sec62.
In the current study, we showed that treatment of different human cancer cells with calmodulin antagonists induced a Sec62-depletion phenotype, including cell migration inhibition and higher sensitivity to Ca2+-driven ER stress. The same effects on tumor cell biology can be expected by treating patients with these substances, which have already been intensively discussed as potential anti-metastatic and anti-proliferative drugs [38–43]. In particular, TFP appears to be a promising candidate for trials in animal models, and in human patients, because it has previously been used as an antipsychotic and antiemetic drug [44, 45]. Treatment with calmodulin antagonists could also provide the means for overcoming problems with treating patients with high levels of Sec62 protein in tumor cells ; here, a personalized therapeutic approach that also targets the SERCA pump using thapsigargin or tissue-specific peptide conjugates of thapsigargin appears to be promising [46–50]. Based on the present results, we propose combined treatment with TFP and targeted thapsigargin as a powerful new strategy for treating patients with SCC of the lung (Figure 7D), which is especially important because the therapeutic options for this malignancy are very limited and increased levels of Sec62 are a significant disadvantage in regard to survival.
The present study describes a new function of Sec62 in regulating the calmodulin-mediated sealing of the Sec61 Ca2+ leakage channel in the ER, which may explain how the up-regulation of SEC62 expression results in reduced survival among lung cancer patients. Furthermore, it provides the first molecular insight into the mechanism of resistance of Sec62-overproducing tumor cells to treatment with thapsigargin. Using calmodulin antagonists, including TFP, we can inhibit cancer cell migration and overcome the problem of Sec62 overproduction in response to thapsigargin, which may also improve the treatment of these cancer entities in future combinatorial therapeutic strategies.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (FOR967, R. Zimmermann) and a donation by Freunde des Universitätsklinikums des Saarlandes (J. Linxweiler and M. Linxweiler).
- Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010, 127: 2893-2917. 10.1002/ijc.25516.View ArticlePubMedGoogle Scholar
- Beaglehole R, Bonita R: Global public health: a scorecard. Lancet. 2008, 372: 1988-1996. 10.1016/S0140-6736(08)61558-5.View ArticlePubMedGoogle Scholar
- Bray FI, Weiderpass E: Lung cancer mortality trends in 36 European countries: secular trends and birth cohort patterns by sex and region 1970–2007. Int J Cancer. 2009, 126: 1454-1466.Google Scholar
- Herbst RS, Heymach JV, Lippman SM: Lung cancer. N Engl J Med. 2008, 359: 1367-1380. 10.1056/NEJMra0802714.View ArticlePubMedGoogle Scholar
- Linxweiler M, Linxweiler J, Barth M, Benedix J, Jung V, Kim YJ, Bohle RM, Zimmermann R, Greiner M: Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer. Am J Pathol. 2012, 180: 473-483. 10.1016/j.ajpath.2011.10.039.View ArticlePubMedGoogle Scholar
- Panzner S, Dreier L, Hartmann E, Kostka S, Rapoport TA: Posttranslational protein transport in yeast reconstituted with a purified complex of Sec proteins and Kar2p. Cell. 1995, 81: 561-570. 10.1016/0092-8674(95)90077-2.View ArticlePubMedGoogle Scholar
- Meyer HA, Grau H, Kraft R, Kostka S, Prehn S, Kalies KU, Hartmann E: Mammalian Sec61 is associated with Sec62 and Sec63. J Biol Chem. 2000, 275: 14550-14557. 10.1074/jbc.275.19.14550.View ArticlePubMedGoogle Scholar
- Tyedmers J, Lerner M, Bies C, Dudek J, Skowronek MH, Haas IG, Heim N, Nastainczyk W, Volkmer J, Zimmermann R: Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes. Proc Natl Acad Sci USA. 2000, 97: 7214-7219. 10.1073/pnas.97.13.7214.View ArticlePubMedPubMed CentralGoogle Scholar
- Lakkaraju AK, Thankappan R, Mary C, Garrison JL, Taunton J, Strub K: Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation. Mol Biol Cell. 2012, 23: 2712-2722. 10.1091/mbc.E12-03-0228.View ArticlePubMedPubMed CentralGoogle Scholar
- Lang S, Benedix J, Fedeles SV, Schorr S, Schirra C, Schauble N, Jalal C, Greiner M, Hassdenteufel S, Tatzelt J, et al: Different effects of Sec61alpha, Sec62 and Sec63 depletion on transport of polypeptides into the endoplasmic reticulum of mammalian cells. J Cell Sci. 2012, 125: 1958-1969. 10.1242/jcs.096727.View ArticlePubMedPubMed CentralGoogle Scholar
- Muller L, Diaz de Escauriaza M, Lajoie P, Theis M, Jung M, Muller A, Burgard C, Greiner M, Snapp EL, Dudek J, Zimmermann R: Evolutionary gain of function for the ER membrane protein Sec62 from yeast to humans. Mol Biol Cell. 2010, 21: 691-703. 10.1091/mbc.E09-08-0730.View ArticlePubMedPubMed CentralGoogle Scholar
- Greiner M, Kreutzer B, Jung V, Grobholz R, Hasenfus A, Stöhr RF, Tornillo L, Dudek J, Stöckle M, Unteregger G, et al: Silencing of the SEC62 gene inhibits migratory and invasive potential of various tumor cells. Int J Cancer. 2011, 128: 2284-2295. 10.1002/ijc.25580.View ArticlePubMedGoogle Scholar
- Greiner M, Kreutzer B, Lang S, Jung V, Adolpho C, Unteregger G, Zimmermann R, Wullich B: Sec62 protein content is crucial for the ER stress tolerance of prostate cancer. Prostate. 2011, 71: 1074-1083. 10.1002/pros.21324.View ArticlePubMedGoogle Scholar
- Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D: IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002, 415: 92-96. 10.1038/415092a.View ArticlePubMedGoogle Scholar
- Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H: ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev. 2002, 16: 1345-1355. 10.1101/gad.992302.View ArticlePubMedPubMed CentralGoogle Scholar
- Huang JB, Kindzelskii AL, Clark AJ, Petty HR: Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion. Cancer Res. 2004, 64: 2482-2489. 10.1158/0008-5472.CAN-03-3501.View ArticlePubMedGoogle Scholar
- Erdmann F, Schauble N, Lang S, Jung M, Honigmann A, Ahmad M, Dudek J, Benedix J, Harsman A, Kopp A, et al: Interaction of calmodulin with Sec61alpha limits Ca2+ leakage from the endoplasmic reticulum. Embo J. 2011, 30: 17-31. 10.1038/emboj.2010.284.View ArticlePubMedGoogle Scholar
- Hilpert K, Winkler DF, Hancock RE: Peptide arrays on cellulose support: SPOT synthesis, a time and cost efficient method for synthesis of large numbers of peptides in a parallel and addressable fashion. Nat Protoc. 2007, 2: 1333-1349. 10.1038/nprot.2007.160.View ArticlePubMedGoogle Scholar
- Aneiros E, Philipp S, Lis A, Freichel M, Cavalie A: Modulation of Ca2+ signaling by Na+/Ca2+ exchangers in mast cells. J Immunol. 2005, 174: 119-130.View ArticlePubMedGoogle Scholar
- Gross SA, Guzman GA, Wissenbach U, Philipp SE, Zhu MX, Bruns D, Cavalie A: TRPC5 is a Ca2 + −activated channel functionally coupled to Ca2 + −selective ion channels. J Biol Chem. 2009, 284: 34423-34432. 10.1074/jbc.M109.018192.View ArticlePubMedPubMed CentralGoogle Scholar
- Lomax RB, Camello C, Van Coppenolle F, Petersen OH, Tepikin AV: Basal and physiological Ca(2+) leak from the endoplasmic reticulum of pancreatic acinar cells. Second messenger-activated channels and translocons. J Biol Chem. 2002, 277: 26479-26485. 10.1074/jbc.M201845200.View ArticlePubMedGoogle Scholar
- Rehberg M, Lepier A, Solchenberger B, Osten P, Blum R: A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum. Cell Calcium. 2008, 44: 386-399. 10.1016/j.ceca.2008.02.002.View ArticlePubMedGoogle Scholar
- Harsman A, Kopp A, Wagner R, Zimmermann R, Jung M: Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates. Channels (Austin). 2011, 5: 293-298. 10.4161/chan.5.4.16160.View ArticleGoogle Scholar
- Lang S, Schauble N, Cavalie A, Zimmermann R: Live cell calcium imaging combined with siRNA mediated gene silencing identifies Ca(2)(+) leak channels in the ER membrane and their regulatory mechanisms. J Vis Exp. 2011Google Scholar
- Chen L, Meng Q, Jing X, Xu P, Luo D: A role for protein kinase C in the regulation of membrane fluidity and Ca2+ flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells. Cell Signal. 2011, 23: 497-505. 10.1016/j.cellsig.2010.11.005.View ArticlePubMedGoogle Scholar
- Nabeshima K, Komada N, Kishi J, Koita H, Inoue T, Hayakawa T, Koono M: TPA-enhanced invasion of Matrigel associated with augmentation of cell motility but not metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1. Int J Cancer. 1993, 55: 974-981. 10.1002/ijc.2910550617.View ArticlePubMedGoogle Scholar
- Lin CW, Shen SC, Chien CC, Yang LY, Shia LT, Chen YC: 12-O-tetradecanoylphorbol-13-acetate-induced invasion/migration of glioblastoma cells through activating PKCalpha/ERK/NF-kappaB-dependent MMP-9 expression. J Cell Physiol. 2010, 225: 472-481. 10.1002/jcp.22226.View ArticlePubMedGoogle Scholar
- Lee J, Ishihara A, Oxford G, Johnson B, Jacobson K: Regulation of cell movement is mediated by stretch-activated calcium channels. Nature. 1999, 400: 382-386. 10.1038/22578.View ArticlePubMedGoogle Scholar
- Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR: Cell migration: integrating signals from front to back. Science. 2003, 302: 1704-1709. 10.1126/science.1092053.View ArticlePubMedGoogle Scholar
- Sjaastad MD, Nelson WJ: Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium. Bioessays. 1997, 19: 47-55. 10.1002/bies.950190109.View ArticlePubMedGoogle Scholar
- Schauble N, Lang S, Jung M, Cappel S, Schorr S, Ulucan O, Linxweiler J, Dudek J, Blum R, Helms V, et al: BiP-mediated closing of the Sec61 channel limits Ca(2+) leakage from the ER. Embo J. 2012, 31: 3282-3296. 10.1038/emboj.2012.189.View ArticlePubMedPubMed CentralGoogle Scholar
- Wittke S, Dunnwald M, Johnsson N: Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex. Mol Biol Cell. 2000, 11: 3859-3871. 10.1091/mbc.11.11.3859.View ArticlePubMedPubMed CentralGoogle Scholar
- Christie RJ, Nishiyama N, Kataoka K: Delivering the code: polyplex carriers for deoxyribonucleic acid and ribonucleic acid interference therapies. Endocrinology. 2009, 151: 466-473.View ArticlePubMedGoogle Scholar
- Jackson AL, Linsley PS: Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nat Rev Drug Discov. 2010, 9: 57-67. 10.1038/nrd3010.View ArticlePubMedGoogle Scholar
- Koehn S, Schaefer HW, Ludwig M, Haag N, Schubert US, Seyfarth L, Imhof D, Markert UR, Poehlmann TG: Cell-specific RNA interference by peptide-inhibited-peptidase-activated siRNAs. J RNAi Gene Silencing. 2010, 6: 422-430.PubMedPubMed CentralGoogle Scholar
- Schmidt C: RNAi momentum fizzles as pharma shifts priorities. Nat Biotechnol. 2011, 29: 93-94. 10.1038/nbt0211-93.View ArticlePubMedGoogle Scholar
- Bonetta L: RNA-based therapeutics: ready for delivery?. Cell. 2009, 136: 581-584. 10.1016/j.cell.2009.02.010.View ArticlePubMedGoogle Scholar
- Coticchia CM, Revankar CM, Deb TB, Dickson RB, Johnson MD: Calmodulin modulates Akt activity in human breast cancer cell lines. Breast Cancer Res Treat. 2009, 115: 545-560. 10.1007/s10549-008-0097-z.View ArticlePubMedGoogle Scholar
- Hwang YP, Jeong HG: Metformin blocks migration and invasion of tumour cells by inhibition of matrix metalloproteinase-9 activation through a calcium and protein kinase Calpha-dependent pathway: phorbol-12-myristate-13-acetate-induced/extracellular signal-regulated kinase/activator protein-1. Br J Pharmacol. 2010, 160: 1195-1211. 10.1111/j.1476-5381.2010.00762.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Jung HJ, Kim JH, Shim JS, Kwon HJ: A novel Ca2+/calmodulin antagonist HBC inhibits angiogenesis and down-regulates hypoxia-inducible factor. J Biol Chem. 2010, 285: 25867-25874. 10.1074/jbc.M110.135632.View ArticlePubMedPubMed CentralGoogle Scholar
- Yuan K, Jing G, Chen J, Liu H, Zhang K, Li Y, Wu H, McDonald JM, Chen Y: Calmodulin mediates Fas-induced FADD-independent survival signaling in pancreatic cancer cells via activation of Src-extracellular signal-regulated kinase (ERK). J Biol Chem. 2011, 286: 24776-24784. 10.1074/jbc.M110.202804.View ArticlePubMedPubMed CentralGoogle Scholar
- Polischouk AG, Holgersson A, Zong D, Stenerlow B, Karlsson HL, Moller L, Viktorsson K, Lewensohn R: The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non small cell lung carcinoma cells to DNA double-strand break induced cell death. Mol Cancer Ther. 2007, 6: 2303-2309. 10.1158/1535-7163.MCT-06-0402.View ArticlePubMedGoogle Scholar
- Satyamoorthy K, Perchellet JP: Modulation by adriamycin, daunomycin, verapamil, and trifluoperazine of the biochemical processes linked to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1989, 49: 5364-5370.PubMedGoogle Scholar
- Carpenter WT, Davis JM: Another view of the history of antipsychotic drug discovery and development. Mol Psychiatry. 2012, 17 (12): 1168-1173. 10.1038/mp.2012.121.View ArticlePubMedGoogle Scholar
- Shen WW: A history of antipsychotic drug development. Compr Psychiatry. 1999, 40: 407-414. 10.1016/S0010-440X(99)90082-2.View ArticlePubMedGoogle Scholar
- Ghantous A, Gali-Muhtasib H, Vuorela H, Saliba NA, Darwiche N: What made sesquiterpene lactones reach cancer clinical trials?. Drug Discov Today. 2010, 15: 668-678. 10.1016/j.drudis.2010.06.002.View ArticlePubMedGoogle Scholar
- Christensen SB, Skytte DM, Denmeade SR, Dionne C, Møller JV, Nissen P, Isaacs JT: A Trojan horse in drug development: targeting of thapsigargins towards prostate cancer cells. Anticancer Agents Med Chem. 2009, 9: 276-294. 10.2174/1871520610909030276.View ArticlePubMedGoogle Scholar
- Huang J-K, Huang C-C, Lu T, Chang H-T, Lin K-L, Tsai J-Y, Liao W-C, Chien J-M, Jan C-R: Effect of MK-886 on Ca2+ level and viability in PC3 human prostate cancer cells. Basic Clin Pharmacol Toxicol. 2009, 104: 441-447. 10.1111/j.1742-7843.2009.00413.x.View ArticlePubMedGoogle Scholar
- Denmeade SR, Isaacs JT: The SERCA pump as a therapeutic target: making a “smart bomb” for prostate cancer. Cancer Biol Ther. 2005, 4: 14-22. 10.4161/cbt.4.1.1505.View ArticlePubMedGoogle Scholar
- Denmeade SR, Mhaka AM, Rosen DM, Brennen WN, Dalrymple S, Dach I, Olesen C, Gurel B, Demarzo AM, Wilding G: Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy. Sci Transl Med. 2012, 4: 140ra186-View ArticleGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/574/prepub
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.