Purpose: In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. Although the contribution of the Gq/11-phospholipase C-β cascade to mitogenic effects in SCLC cells is well established, the relevance of G12/13 signaling is still elusive. In other tumor entities, G12/13 activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G12/13-dependent signaling in SCLC.

Experimental Design: We used small hairpin RNA–mediated targeting of Gα12, Gα13, or both in H69 and H209 cells and analyzed the effects of Gα12 and/or Gα13 knockdown on tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase (MAPK) activation.

Results: Lentiviral expression of small hairpin RNAs resulted in robust and specific Gα12 and Gα13 knockdown as well as markedly inhibited proliferation, colony formation, and bradykinin-promoted stimulation of cell growth. Analyzing the activation status of all three major MAPK families revealed nonredundant functions of Gα12 and Gα13 in SCLC and a marked p42/p44 activation upon Gα12/Gα13 knockdown. In a s.c. tumor xenograft mouse model, Gα12 or Gα13 downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, Gα12/Gα13 double knockdown completely abolished H69 tumorigenicity in mice.

Conclusions:12 and Gα13 exert a complex pattern of nonredundant effects in SCLC, and in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G12/13 signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment. Clin Cancer Res; 16(5); 1402–15

Translational Relevance

Small cell lung cancer (SCLC) displays the most aggressive clinical course among all common types of pulmonary tumors, and the poor survival rates emphasize the need for novel treatment strategies based on a deeper understanding of the molecular events underlying SCLC tumorigenesis and tumor progression. Various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins; however, the relevance of G12/13 signaling is still elusive. In this study based on small hairpin RNA (shRNA)–mediated knockdown of Gα12 and/or Gα13, we investigate the role of G12/13-dependent signaling on SCLC tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase activation. We show that Gα12 and Gα13 exert a complex pattern of nonredundant effects in SCLC and show that, in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G12/13 signaling. Strikingly, Gα12/Gα13 double knockdown completely abolishes H69 tumorigenicity in mice. Although the inhibition of single G protein–coupled receptors or G protein–coupled receptor agonists may be insufficient for treatment, we show that the parallel knockdown of Gα12 and Gα13 as point of convergence may provide a promising new avenue in SCLC treatment.

Members of the G protein–coupled receptor (GPCR) superfamily are involved in the regulation of virtually all cellular processes, and G protein–dependent signaling pathways have been shown to play a pivotal role in the pathogenesis of tumors. Heterotrimeric G proteins are composed of a guanine nucleotide binding α-subunit and a βγ dimer. Based on sequence homologies, four families of G protein-α subunits can be distinguished, i.e., GS, Gi/o, Gq/11, and G12/13 (13). The constitutive activation of GPCRs or G proteins has been shown in various tumor entities and is implicated in cellular proliferation, regulation of apoptosis, enhanced cell motility and invasiveness, metastatic potential of tumor cells, as well as angiogenic effects (4, 5). Although the diversity of G protein–dependent functions in tumor cells is mirrored by the fact that G proteins of all four families have been identified as potential oncogenes, the G12/13 family has attracted particular interest in cancer research because its members have been described to promote the growth and oncogenic transformation of murine fibroblasts (6, 7) and G12/13 proteins have been invoked in tumorigenesis (8). Gα12 was first described as gep oncogene in soft tissue sarcomas. The expression of wild-type (WT) Gα12 in fibroblasts turned out to be sufficient for cell transformation (6). More recently, a role of G12/13 signaling has been shown in breast and prostate cancer (9, 10). In these tumors, G12/13-dependent signaling was shown to regulate tissue invasiveness and metastatic capacity, but not proliferation of tumor cells. This observation is in line with the well-characterized ability of G12/13 to activate monomeric GTPases of the Rho family and, thus, to control cytoskeletal proteins and cellular motility (11).

Lung cancer accounts for over 200,000 new cases and over 160,000 deaths per year in the United States. Small cell lung cancer (SCLC) displays the most aggressive clinical course among all types of pulmonary tumors. Although current chemotherapy regimen clearly show clinical benefit, the overall survival after 5 years is still only 7% to 12% (12), and patients with SCLC tend to develop distant metastases. This situation clearly emphasizes the need for novel treatment strategies based on a deeper understanding of the molecular events underlying SCLC tumorigenesis and tumor progression.

SCLC cells have been shown to secrete a plethora of GPCR ligands, e.g., acetyl choline or neuropeptides such as bradykinin, bombesin/gastrin-releasing peptide, or galanin, which act as principal mitogenic stimuli (13, 14). Owing to the critical role of G protein signaling for the control of proliferation in SCLC cells, many efforts have been made to use blockers of mitogenic GPCRs expressed in SCLC as therapeutic tools (1519). However, the redundance of multiple autocrine loops in SCLCs promoting the activation of numerous distinct GPCRs limits the therapeutic efficacy of this approach. Thus, points of convergence of mitogenic GPCR signaling in SCLC cells could turn out as more promising therapeutic targets. The GPCRs that regulate proliferation in SCLC cells couple to Gq/11 and G12/13 proteins, leading to the parallel activation of the Gq/11-phospholipase C-Ras-extracellular signal-regulated kinase (ERK) 1/2 and the G12/13-Rho signaling pathways (20). Although activation of Gq/11 and phospholipase Cβ causes an increase in intracellular calcium ([Ca2+]i), which is necessary and sufficient to activate the Ras-Raf-ERK1/2 cascade in SCLC cells (21), G12/13 signaling in SCLC cells has been implicated in the regulation of cell motility and metastasis (22, 23) and in the induction of proapoptotic pathways through the activation of c-Jun-NH2-kinase (24). However, the relative contribution of Gq/11 and G12/13-dependent signaling cascades to the cellular phenotype of SCLC cells is presently unknown (21). Furthermore, previous studies suggest different biological effects of G12 and G13 (25), thus emphasizing the need to assess the functional relevance of both G proteins separately.

In this article, we aimed at the selective disruption of G12 and G13 function to decipher the role of these proteins for oncogenic signaling in SCLC cells. To this end, we used RNAi-mediated downregulation of the α-subunits of G12 and G13 (Gα12 and Gα13), alone or in combination, by infection of H69 and H209 SCLC cells with shRNA constructs using a lentiviral system. Single targeting revealed profound antiproliferative effects in response to Gα12 or Gα13 downregulation. The analysis of the activation status of all three major mitogen-activated protein kinase (MAPK) families revealed nonredundant functions of Gα12 and Gα13 in SCLC as well as a marked p42/p44 activation upon Gα12/Gα13 knockdown. In a s.c. tumor xenograft mouse model, Gα12 or Gα13 single knockdown led to a markedly reduced tumor growth based on decreased tumor cell proliferation. More importantly, Gα12/Gα13 double targeting resulted in complete abolishment of tumor growth. Thus, we show for the first time that both Gα12 and Gα13 play a critical and nonredundant role in SCLC proliferation in vitro and in vivo, and that selectively interfering with G12 and G13 signaling, e.g., through RNAi-mediated therapeutic blockage of both G proteins, may provide a promising new avenue in SCLC treatment.

Cell lines, viral shRNA constructs, and stable transfection

Lung cancer cell lines were obtained from the American Type Culture Collection and were cultivated under standard conditions (37°C, 5% CO2) in RPMI (PAA) supplemented with 10% FCS unless indicated otherwise. Oligonucleotides for the generation of shRNA were designed according to established rules (26, 27), and the DNA sequences detailed in Supplementary Table S1 were chemically synthesized (MWG Biotech). Using the BglII and HindIII restriction sites, the oligonucleotides were cloned into the pSUPER vector and correct insertion was controlled by sequencing. Subsequently, the H1-cassettes of the pSUPER vectors were cloned into the lentivirus vector pLVTHM using ClaI and BamHI. To generate recombinant viruses, 20 μg/10-cm dish of the cis-vector pLV-12-1, pLV-12-2, pLV-12-3 (Gα12-specific shRNAs), pLV-13-1, pLV-13-2, pLV-13-3 (Gα13-specific shRNAs), pLV-UR-1, pLV-UR-2, or pLV-UR-3 (control shRNAs) were cotransfected into HEK cells together with 15 μg psPAX2 (“trans plasmid”) and with 6 μg pMD2G (VSV-G) using the calcium phosphate precipitation method. Six to 8 h after transfection, the medium was changed, and 2 d later, the supernatant was collected and sterile filtered. Viruses were purified and concentrated using sucrose sedimentation (20% sucrose in TBS, 26,000 rpm, 2 h) and were resuspended in complete RPMI. The viral titer was determined by serial dilution in HEK cells. H69 and H209 cells were transduced with 20 transducing units of recombinant virus.

RNA preparation and quantitative reverse transcription-PCR

Total RNA from tumor cells or homogenized tissues was isolated using the Tri reagent (PEQLAB) according to the manufacturer's protocol. For tissue homogenization, tissues were mixed with 1 mL Tri reagent and homogenized before RNA preparation. Reverse transcription was done using the RevertAid H Minus First Strand cDNA Synthesis kit from Fermentas as follows: 2 μg total RNA and 1 μL random hexamer primer (0.2 μg/μL) were diluted in DEPC-treated water to a final volume of 11 μL, incubated at 70°C for 5 min and chilled on ice before adding 4 μL 5× reaction buffer, 0.5 μL RNase inhibitor (20 u/μL), 2 μL 10 mmol/L deoxynucleotide triphosphate mix, and 1.5 μL DEPC-treated water. After incubation at 25°C for 5 min, 1 μL reverse transcriptase (200 u/μL) was added, and the mixture was incubated for 10 min under the same conditions and for 60 min at 42°C, before stopping the reaction by heating at 70°C for 10 min and chilling on ice. Quantitative PCR was done in a LightCycler from Roche using the Absolute QPCR SYBRGREEN Capillary Mix (Abgene) according to the manufacturer's protocol with 4 μL cDNA (diluted 1:100), 1 μL primers (5 μmol/L each), and 5 μL SYBR Green master mix. A preincubation for 15 min at 95°C was followed by 55 amplification cycles: 10 s at 95°C, 10 s at 55°C, and 10 s at 72°C. The melting curve for PCR product analysis was determined by rapid cooling down from 95°C to 65°C, and incubation at 65°C for 15 s before heating to 95°C. To normalize for equal mRNA/cDNA amounts, PCRs with Gα12- or Gα13-specific and with actin-specific primer sets were always run in parallel for each sample, and Gα12/Gα13 levels were determined by the formula 2CP(G protein) /2CP(actin) in which CP is the cycle number at the crossing point (0.3).

Growth assays

For proliferation studies, cell lines were plated into 96-well plates at 2,000 to 4,000 cells per well and 5 wells per time point, and cultivated in RPMI/10% FCS in a humidified incubator under standard conditions. The numbers of viable cells were assessed using a colorimetric assay according to the manufacturer's protocol (Cell Proliferation Reagent WST-1, Roche Molecular Biochemicals), or were counted microscopically using a Neubauer chamber. Where indicated, 1 μmol/L ERK 1/2 inhibitor U0126 (Promega) or 10 μmol/L Rho kinase inhibitor Y27632 (Sigma, dissolved in DMSO) was added with appropriate volumes of DMSO serving as negative controls. The role of Rho in cellular proliferation was also addressed by using a clostridial C3 fusion protein together with the activated C2II binding component of C. botulinum C2 toxin (both were kindly provided by Holger Barth, Ulm, Germany). For this purpose, the cells were treated with the fusion toxin at 200 ng/mL, a concentration reliably inactivating Rho proteins (see e.g., ref. 28). For bradykinin stimulation experiments, a 10-mmol/L bradykinin stock solution, prepared in 5% acetic acid, was diluted in PBS and added to the wells containing RPMI/10% FCS to a final concentration of 100 nmol/L, with the appropriate negative controls.

To study proliferation of the cell lines in a gel matrix, soft agar assays were carried out essentially as described in ref. (29). Briefly, 100,000 cells in 0.35% agar (Bacto Agar, Becton Dickinson) were layered on top of 1 mL of a solidified 0.6% agar layer in a 35-mm dish. Growth media with 10% FCS were included in both layers. Colonies of >50 μm in diameter were counted after 2 to 3 wk of incubation by an image analyzer or by at least two independent blinded investigators.

Tumor growth in nude mice

WT or stably infected H69 cells (3 × 106) in 150 μL PBS were injected s.c. into both flanks of athymic nude mice (nu/nu) with 10 tumors per cell line. When solid tumors became visible after 1 wk, tumor growth was monitored every 2 to 3 d as indicated in the figure, and tumor sizes were determined from the product of the perpendicular diameters of the tumors. After 4 wk, mice were sacrificed and tumors were removed. Three pieces of each tumor representing approximately half of the tumor mass were immediately fixed in 10% paraformaldehyde for paraffin embedding and the other half was shock frozen in liquid nitrogen for RNA extraction. In the case of H209 cell lines, 5 × 106 cells were injected, and after 4 wk, mice with well-established tumors below 100 mm3 were selected for the subsequent monitoring of tumor growth rates as described above.

Immunohistochemistry

Paraffin sections of each tumor contained different pieces of the tumor mass. Immunostaining was done essentially as previously described (30). Briefly, after deparaffinization with xylene and rehydration with graded alcohols, sections were incubated in 10 mmol/L citrate buffer (pH 7.4) at 90°C for 20 min, endogenous peroxidases were inactivated with 0.3% hydrogen peroxide at 4°C for 20 min, and slides were washed thrice with PBS/0.1% Tween 20. After blocking with 10% normal goat serum or normal horse serum in PBS/0.1% Tween 20/2% bovine serum albumin for 1 h at room temperature, the slides were incubated with the antibodies anti-G12 (Santa Cruz, 1:200), anti-G13 (Santa Cruz, 1:200), or anti–proliferating cell nuclear antigen (PCNA; DAKO, 1:200) in PBS/0.1% Tween 20 at 4°C overnight in a wet chamber. After washing, a 1:500 diluted, biotinylated goat anti–(rabbit-IgG) or horse anti–(mouse-IgG) antibody (Vector Laboratories; 1:500) was applied as the secondary antibody for 1 h before washing. Immunoreactivity on the sections was visualized using a streptavidin-biotin-peroxidase complex (ABC kit, Vector Laboratories) according to the manufacturer's instructions, with diaminobenzidine as peroxidase substrate (brown). PCNA-stained sections were counterstained with hematoxylin, mounted, and analyzed light microscopically by three blinded observers. For the quantitation of G12 and G13 levels, the counterstain was omitted, and upon mounting, slides were light microscopically scanned and electronically analyzed for intensities of brown staining (TILL Photonics and TILL Vision).

Antibody arrays

To analyze the phosphorylation status of various MAPKs in control versus Gα12 or control versus Gα13 knockdown cells, proteome profiler antibody arrays (R&D) were used according to the manufacturer's instructions. Briefly, after preincubation in Array buffer 1, arrays were incubated overnight at 4°C in 250 μL or the respective cell lysate in Lysis buffer 6/1,250 μL Array buffer 1. After washing, arrays were incubated in a 1:100 dilution of the Detection Antibody Cocktail Concentrate for 2 h at room temperature and washed again. Bound antibodies were detected using Streptavidin-horseradish peroxidase (1:2,000, 30 min at room temperature), and after another washing step, signals were visualized by incubation of the arrays in the LumiGLO chemiluminescent reagent and parallel film exposure (Hyperfilm, Amersham). To accurately determine signals of different intensities, films were exposed between 5 s and 10 min, and signals above the background and below the film saturation were scanned and quantitated using the ImageJ software.

Western blots

For Western blot analysis, SCLC cells were grown in six-well plates coated with poly-l-lysine (6 × 105 cells per well) for 24 h. Cells were washed with ice-cold PBS and lysed in 200 μL of lysis buffer [125 mmol/L Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 50 μg/mL bromphenol blue]. Proteins were separated by SDS-PAGE (9% gel) and electroblotted onto Hybond C Extra membranes. Membranes were incubated in Rotiblock (Roth) for 1 h to saturate nonspecific binding sites, washed in PBS, and incubated with the respective primary antibody at 4°C overnight. Phosphorylated p44/42 MAPK was detected using a phospho-specific anti-p44/42 MAPK mouse monoclonal antibody (Santa Cruz Biotechnology, 1:500). Phosphorylated MYPT1 (Threonine 853) was detected using a phospho-specific monoclonal antibody from a rabbit (New England Biolabs, 1:1,000). Antibodies against the Gα subunits of G12 and G13 were from Santa Cruz (1:500). Reprobing for loading controls was done using an anti-p44/42 MAPK mouse monoclonal antibody detecting total (phosphorylated and unphosphorylated) ERK. Western blots were developed by chemiluminescence as described above.

Data analysis

For statistical analysis, Student's unpaired, two-sided t test was used for comparisons between data sets. Unless indicated otherwise, bars represent the means of at least three independent experiments. For cost reasons, the antibody array was done only once, and representative results were subsequently confirmed by Western blotting in independent experiments with fresh cell lysates. The immunohistochemical analysis was done in all tumor xenografts (n = 10) with at least three fields per section being randomly selected for microscopic quantitation. Error bars in the figures represent SDs.

Determination of Gα12 and Gα13 expression levels in lung cancer cell lines

12 and/or Gα13 expression levels were determined by quantitative reverse transcription-PCR (RT-PCR) in the classic SCLC lines H69, H209, H510, H146, and H187. Additionally, the non-SCLC lines A549, HAE, and HBE as well as the nonclassic SCLC line H82 were included. All cell lines were positive for Gα12 (Fig. 1A) and Gα13 (Fig. 1B), with approximately 2- to 2.5-fold differences in Gα12 expression levels independent of the origin of the cell lines (SCLC or non-SCLC). Gα13 expression showed larger variations with ∼15-fold differences between the highest and the lowest Gα13-expressing cell line. Notably, no correlation was found between Gα12 and Gα13 expression levels. For subsequent experiments, we selected the SCLC cell lines H69, which showed the highest expression levels of both Gα12 and Gα13, and H209 with lower Gα12 and high Gα13 expression.

Fig. 1.

Expression levels of Gα12 (A) and Gα13 (B) in various SCLC and NSCLC cell lines, as determined by quantitative RT-PCR. Values are normalized for actin as described in Materials and Methods and presented in arbitrary units.

Fig. 1.

Expression levels of Gα12 (A) and Gα13 (B) in various SCLC and NSCLC cell lines, as determined by quantitative RT-PCR. Values are normalized for actin as described in Materials and Methods and presented in arbitrary units.

Close modal

RNAi-mediated depletion of Gα12 and Gα13 in SCLC cells

Initial experiments revealed that nonviral transfection efficacies in H69 and H209 cells were extremely poor. Despite the fact that the use of a recently described low-molecular weight polyethylenimine (31) resulted in somewhat higher efficacies in DNA uptake and expression compared with several other transfection agents, we chose a viral approach for expressing shRNA constructs to induce RNAi-mediated Gα12 and/or Gα13 knockdown. shRNA expression plasmids were constructed by cloning the DNA oligonucleotides detailed in Supplementary Table S1 into an enhanced green fluorescent protein (EGFP) expression vector. Upon viral infection with these constructs, transcription is expected to result in a partially double-stranded RNA sequence (boxed in Supplementary Table S1) being recognized by Dicer and thus qualifying for the specific induction of RNA interference according to the rules of Reynolds et al. and Elbashir et al. (26, 27). Cellular EGFP expression upon viral infection allowed the simultaneous assessment of DNA uptake efficacy. The morphologic analysis of the cells by fluorescence microscopy revealed an infection efficiency of ∼100% (Fig. 2A).

Fig. 2.

A, viral infection of SCLC cells with shRNA constructs, as determined by EGFP expression in fluorescence microscopy (left). The comparison with bright-field microscopy (right) reveals an ∼100% efficacy. B, upon Gα12 or Gα13 knockdown in H69 cells, levels of Gα11 and Gαq remain unchanged. C and D, specific targeting efficacies of Gα12 shRNA (left) and Gα13 shRNA (right) in H69 (C) and H209 (D) cells (WT, 100%). E, Western blots for Gα12 and Gα13 in H69 cells reveal the specific knockdown of Gα12 and Gα13 protein levels (*).

Fig. 2.

A, viral infection of SCLC cells with shRNA constructs, as determined by EGFP expression in fluorescence microscopy (left). The comparison with bright-field microscopy (right) reveals an ∼100% efficacy. B, upon Gα12 or Gα13 knockdown in H69 cells, levels of Gα11 and Gαq remain unchanged. C and D, specific targeting efficacies of Gα12 shRNA (left) and Gα13 shRNA (right) in H69 (C) and H209 (D) cells (WT, 100%). E, Western blots for Gα12 and Gα13 in H69 cells reveal the specific knockdown of Gα12 and Gα13 protein levels (*).

Close modal

H69 cells virally infected with shRNA constructs were analyzed by quantitative RT-PCR to evaluate the depletion of Gα12 and/or Gα13. To avoid nonspecific small interfering RNA effects, which have been previously described and may obscure any gene-specific biological effects (see, e.g., ref. 35 for review), three different constructs per target gene and three control shRNAs (Supplementary Table S1) were used and tested independently for targeting efficacy and antiproliferative effects. Quantitative RT-PCR revealed that Gα12-targeting shRNAs 12-1 and 12-2 were similarly efficient, as well as all three Gα13-targeting shRNAs. Proliferation assays showed a concomitant reduction in cell proliferation (see below). Based on the targeting efficacies and antiproliferating effects, constructs 12-1 and 13-1 were selected for subsequent experiments.

The Gα12-targeting shRNA downregulated Gα12 mRNA in H69 and H209 cells by >50% and ∼90%, respectively, when compared with control-infected or WT (i.e., parental, nontransduced) cells (Fig. 2C and D, left). Gα13 levels remained unchanged, proving the specificity of gene targeting. Likewise, Gα13-specific shRNAs resulted in a profound 50% to 85% reduction of Gα13 mRNA levels in both cell lines, without affecting Gα12 (Fig. 2C and D, right). Gene-specific and similar targeting efficacies were also observed upon double infection with both shRNA constructs (Fig. 2C and D). Likewise, specific knockdown was observed on the protein level. Western blots revealed the absence or near absence of Gα12 or Gα13 bands, respectively, upon single and double knockdown (Fig. 2E, *). Because these bands were below the limit of detection, no quantitation was possible. In contrast, mRNA levels of other G proteins, Gα11 and Gαq, remained unchanged upon Gα12 or Gα13 knockdown (Fig. 2B). The fact that highly reproducible data were obtained when using the various Gα12- or Gα13-specific versus unrelated shRNA constructs confirmed the suitability of our approach for the specific depletion of Gα12 and Gα13, and the correlation between knockdown efficiencies and antiproliferative effects suggests the absence of nonspecific off-target effects.

12/Gα13 dependence of SCLC cell proliferation

Monitoring the cell proliferation of H69 cells infected with Gα12 and/or Gα13-shRNA constructs over several days revealed a markedly reduced proliferative capacity upon Gα12 or Gα13 depletion. More specifically, Gα13 targeting led to a ∼50% reduction in cell number, whereas upon Gα12 depletion, a more profound inhibitory effect (>60%) on cell proliferation was observed compared with WT or control-infected cells (Fig. 3A). This effect was observed for all constructs that showed gene targeting efficacy, i.e., shRNAs 12-1, 12-2, 13-1, 13-2, and 13-3, but not for shRNA 12-3 with no targeting and no antiproliferative activities, thus confirming the specificity of the active shRNAs and suggesting the absence of nonspecific effects. Furthermore, the proliferation data showed identical results of WT cells and those infected with unrelated control shRNA, thereby excluding nonspecific effects intrinsically due to viral infection (Fig. 3A). Double targeting, i.e., the combined infection with both constructs, resulted in antiproliferative effects similar to the results after single targeting (Fig. 3A). Thus, the knockdown of both Gα12 and Gα13 did not produce a synergistic growth-inhibiting effect in vitro. Next, we performed soft agar assays that more closely resemble the in vivo situation. In H69 cells, colony formation was markedly reduced by approximately 50% to 65% upon Gα12 or Gα13 targeting. Combined infection with shRNA constructs targeting both gene products yielded similar results (Fig. 3B). These results were confirmed in the second SCLC cell line investigated, H209 cells. While due to poor colony formation, soft agar assays could not be done, WST-1-based proliferation assays revealed proliferation rates that were profoundly reduced by approximately 50% to 65% upon viral Gα12 or Gα13 shRNA infection (Fig. 3C). Double targeting of both Gα12 and Gα13 led to comparable results, again reflecting the lack of an additive antiproliferative effect upon the knockdown of both proteins (Fig. 3C).

Fig. 3.

Antiproliferative effects of shRNA-mediated downregulation of Gα12 and Gα13 in H69 and H209 SCLC cells. A, the compilation of several proliferation assays (day 1: time point of plating of the stable cell lines; day 6: cell densities 5 d after plating) establishes the reduced proliferation of H69 cells upon Gα12 or Gα13 targeting. No additive effects upon double targeting are observed. B, soft agar assay showing decreased colony formation of H69 cells upon shRNA-mediated Gα12 or Gα13 targeting. C, antiproliferative effects of shRNA-mediated downregulation of Gα12 and Gα13 in H209 SCLC cells. The compilation of several proliferation assays shows the reduced proliferation upon Gα12 or Gα13 targeting. No additive effects upon double targeting are observed.

Fig. 3.

Antiproliferative effects of shRNA-mediated downregulation of Gα12 and Gα13 in H69 and H209 SCLC cells. A, the compilation of several proliferation assays (day 1: time point of plating of the stable cell lines; day 6: cell densities 5 d after plating) establishes the reduced proliferation of H69 cells upon Gα12 or Gα13 targeting. No additive effects upon double targeting are observed. B, soft agar assay showing decreased colony formation of H69 cells upon shRNA-mediated Gα12 or Gα13 targeting. C, antiproliferative effects of shRNA-mediated downregulation of Gα12 and Gα13 in H209 SCLC cells. The compilation of several proliferation assays shows the reduced proliferation upon Gα12 or Gα13 targeting. No additive effects upon double targeting are observed.

Close modal

G12/G13 proteins have previously been shown to signal through Rho kinases. Thus, decreased Rho kinase activity may entail antiproliferative effects. To test this possibility, H69 and H209 WT cells were treated with the Rho kinase inhibitor Y27632 (10 μmol/L) and cell proliferation was compared with nontreated WT cells. As shown in Fig. 3A and B, no differences were observed in H69 and H209 cells, indicating that Rho kinase activation does not play a major role for the antiproliferative effects emanating from Gα12 and/or Gα13 targeting. To confirm the efficacy of the Rho kinase inhibitor Y27632 in our cells, we determined the phosphorylation of MYPT1, which is the regulatory unit of myosine phosphatase. Upon activation of Rho, MYPT1 is phosphorylated by Rho kinase at Threonine 696, leading to an inhibition of the myosine phosphatase holoenzyme (see ref. 32 for review). Consequently, treatment of H69 WT cells with Y27632 (10 μmol/L, 1 hour) completely abolished basal MYPT1 phosphorylation (Supplementary Fig. S1, lanes 1 and 2). Interestingly, a similarly pronounced reduction of MYPT1 phosphorylation in H69 cells independent of the presence or absence of inhibitor was observed upon the shRNA-mediated downregulation of Gα12/Gα13 (Supplementary Fig. S1, lanes 3 and 4 versus lane 1), most probably reflecting the impaired Rho/Rho kinase signaling following Gα12/Gα13 knockdown.

In line with the observed absence of an effect of Rho kinases on H69 cell proliferation, the inactivation of Rho proteins by treatment of the cells with the clostridial fusion toxin C3-FT together with C2IIa (200 ng/mL each) did not affect cell proliferation (Fig. 4C).

To test whether the growth-inhibiting effects of Gα12 and Gα13 knockdown also occurred upon the stimulation of G12/13 coupled receptors, we evaluated the proliferation of SCLC cells following treatment with bradykinin. Stimulation with bradykinin leads to the parallel activation of Gq/11 and G12/13 signaling, and its mitogenic effects in SCLC cells are well established (21). Indeed, bradykinin treatment of H69 cells resulted in the stimulation of cell proliferation. However, this effect was largely reduced upon Gα12 or Gα13 targeting and was almost completely abolished upon Gα12/Gα13 double knockdown (Fig. 4D). These data strongly support the notion that Gα12 and Gα13 are involved in bradykinin-mediated stimulation of cell proliferation.

Fig. 4.

H69 (A) and H209 (B) proliferation is independent of Rho/Rho kinase as shown by identical proliferation rates in the presence (black columns) or absence (white columns) of the Rho kinase inhibitor Y27632. C, likewise, no differences in proliferation are observed upon treatment (black columns) with a cell-permeable variant of clostridial C3 transferase, which inhibits Rho proteins, and with clostridial toxin B inhibiting Rho, Rac, and Cdc42. D, bradykinin-mediated stimulation of the proliferation of H69 cells grown in the presence of 10% FCS is inhibited upon Gα12 or Gα13 targeting. Double knockdown of both Gα12 and Gα13 leads to the almost complete abolishment of bradykinin stimulation.

Fig. 4.

H69 (A) and H209 (B) proliferation is independent of Rho/Rho kinase as shown by identical proliferation rates in the presence (black columns) or absence (white columns) of the Rho kinase inhibitor Y27632. C, likewise, no differences in proliferation are observed upon treatment (black columns) with a cell-permeable variant of clostridial C3 transferase, which inhibits Rho proteins, and with clostridial toxin B inhibiting Rho, Rac, and Cdc42. D, bradykinin-mediated stimulation of the proliferation of H69 cells grown in the presence of 10% FCS is inhibited upon Gα12 or Gα13 targeting. Double knockdown of both Gα12 and Gα13 leads to the almost complete abolishment of bradykinin stimulation.

Close modal

To ensure that Gq/11-dependent signaling was still functional upon Gα12 or Gα13 knockdown, we determined intracellular Ca2+ levels in H69 cells. These Ca2+ measurements revealed that downregulation of Gα12 and/or Gα13 had no effect on basal intracellular Ca2+ levels (Supplementary Fig. S2). In addition, reduced Gα12 and/or Gα13 levels did not affect bradykinin-promoted increase in Ca2+ (Supplementary Fig. S3). Thus, the observed growth inhibition upon Gα12 or Gα13 knockdown is not mediated through alterations in Ca2+ signaling.

In vivo tumor growth of G12/G13-depleted SCLC cells

For the analysis of in vivo tumor growth, H69 cells virally infected with shRNAs as indicated in Fig. 5 were injected s.c. into athymic nude mice. While after an initial approximately 7- to 10-day lag phase, WT and control-infected cells rapidly formed nodules eventually resulting in large tumors, the growth of Gα12 or Gα13-depleted tumors was dramatically reduced by >50%. This decrease was statistically significant at day 19. After 4 weeks, the mean volume of the tumors infected with Gα12- or Gα13-specific shRNA amounted to only approximately 20% to 30% of control (Fig. 5). Antitumorigenic effects were also observed when investigating the initial tumor take, i.e., the total number of tumors formed after s.c. cell injection (Table 1). Although no differences were observed between WT, control, and Gα13 shRNA-infected cells, tumorigenicity was markedly reduced upon Gα12 targeting, thus suggesting different biological consequences of Gα12 and Gα13 depletion. Even more striking was the antitumorigenic effect upon the double targeting of Gα12 and Gα13, which completely abolished tumor growth, thus underscoring an additive role of both proteins in tumors in vivo. Comparable antitumor effects upon Gα12 or Gα13 single knockdown were obtained in H209 xenografts. Here, a generally poor tumor take was observed even in WT cells, which is in line with their poor colony formation properties in soft agar assays as mentioned above. This effect in combination with considerably larger variations in the initial tumor sizes prevented an analysis similarly accurate as in the H69 xenografts. Still, when tumors, which were well established and within the same size range below 100 mm3 after 4 weeks, were analyzed for their further growth rates, a ∼25% decrease in tumor growth upon Gα13 knockdown was observed. As seen in the previous in vitro experiments, Gα12 knockdown resulted in more profound antitumor effects, which were also observed upon double knockdown (∼65% reduction of tumor growth in both cell lines; data not shown).

Fig. 5.

shRNA-mediated double targeting of Gα12 and Gα13 abolishes tumor growth in a s.c. SCLC tumor xenograft mouse model. Single targeting of Gα12 or Gα13 results in reduced tumor growth compared with WT or control-infected H69 cells, with double targeting revealing an additive effect.

Fig. 5.

shRNA-mediated double targeting of Gα12 and Gα13 abolishes tumor growth in a s.c. SCLC tumor xenograft mouse model. Single targeting of Gα12 or Gα13 results in reduced tumor growth compared with WT or control-infected H69 cells, with double targeting revealing an additive effect.

Close modal
Table 1.

Tumor takes upon injection of the infected cells, i.e., percentage of visible tumors of 10 s.c. injections per group, reveal a complete loss of tumorigenicity upon Gα12/Gα13 double targeting

shRNAPercentage of visible tumors
WT 70 
G12 20 
G13 70 
G12 + 13 
Unrelated 90 
shRNAPercentage of visible tumors
WT 70 
G12 20 
G13 70 
G12 + 13 
Unrelated 90 

For further analysis, the H69 xenografts were selected due to the larger number of tumor samples. Mice were sacrificed at day 28 and Gα12/Gα13 expression was determined on mRNA and protein levels. Quantitative RT-PCR revealed an ∼40% reduction of Gα12 or Gα13 upon specific, shRNA-mediated targeting compared with the control groups (WT and unrelated shRNA infection), whereas the mRNA of the nontargeted protein (Gα13 or Gα12, respectively) remained unchanged (Fig. 6A and C). Because double targeting led to the complete abolishment of tumor growth, this group could not be included in the analysis. Concomitantly, the reduced mRNA levels translated into decreased Gα12/Gα13 protein levels as determined by immunohistochemistry. For quantification, paraffin sections were immunohistochemically stained for Gα12 or Gα13 and microscopically scanned under low magnification without hematoxilin counterstain (see Fig. 6B and D, right panel for representative areas). The subsequent determination of the brown staining intensities were done essentially as previously described (33) and revealed a robust (>70%) reduction of Gα12 or Gα13 protein levels, whereas expression in the control groups as well as levels of the nontargeted G protein remained unchanged (Fig. 6B and D, left). These results confirmed that the antitumorigenic effects were based on the specific downregulation of Gα12/Gα13. Finally, because our in vitro experiments indicated proliferative effects of Gα12/Gα13 expression in SCLC, tumor sections were stained for PCNA to determine the degree of proliferation in the different groups. Microscopic assessment of the percentage of proliferating cells revealed a slight but statistically significant decrease in cell proliferation by ∼30% upon Gα12 or Gα13 targeting (Fig. 6E), indicating that observed differences in tumor growth are based on reduced tumor cell proliferation.

Fig. 6.

Analysis of s.c. tumor xenografts form the SCLC mouse model. A to D, analysis of Gα12 (A and B) and Gα13 (C and D) expression on mRNA (A and C) and protein (B and D) levels in tumor xenografts derived from H69 cells stably transduced as indicated on the X-axis. B and D, right, fields representative for brown intensities (shown here in black and white) after immunohistochemical staining for Gα12 (B) or Gα13 (D), for better quantitation without hematoxylin counterstaining. E, reduced tumor cell proliferation upon Gα12 or Gα13 knockdown as shown by the immunohistochemical staining of the proliferation marker PCNA; left, quantitation of positive nuclei (brown); right, representative examples; positive nuclei appear as dark spots in this black and white reproduction.

Fig. 6.

Analysis of s.c. tumor xenografts form the SCLC mouse model. A to D, analysis of Gα12 (A and B) and Gα13 (C and D) expression on mRNA (A and C) and protein (B and D) levels in tumor xenografts derived from H69 cells stably transduced as indicated on the X-axis. B and D, right, fields representative for brown intensities (shown here in black and white) after immunohistochemical staining for Gα12 (B) or Gα13 (D), for better quantitation without hematoxylin counterstaining. E, reduced tumor cell proliferation upon Gα12 or Gα13 knockdown as shown by the immunohistochemical staining of the proliferation marker PCNA; left, quantitation of positive nuclei (brown); right, representative examples; positive nuclei appear as dark spots in this black and white reproduction.

Close modal

Differential activation of MAPK pathways

To assess the status of activation of various MAPK signal transduction pathways upon stable knockdown of Gα12 or Gα13, antibody arrays were done for H69 cells stably transfected with Gα12 shRNA or with Gα13 shRNA versus control shRNA, monitoring the phosphorylation of key signaling proteins. Signals showed considerable differences with regard to intensities, depending on the analyte. Therefore, the accurate detection of chemiluminescence relied on several different exposures (see Fig. 7 A and B, lower panel for representative examples) with signals close to the background or above the film saturation being excluded from quantitation.

Fig. 7.

Antibody arrays for the assessment of changes in the phosphorylation of various MAPK signal transduction pathways upon stable knockdown of Gα12 or Gα13. Arrays were probed with lysates from H69 cells stably transfected with control shRNA versus Gα12 shRNA (A) or with control shRNA versus Gα13 shRNA (B). Bottom, two representative examples of exposures per array used for quantitation (signals in the uppermost rows: loading controls; all other signals: analyte spots as dotted by the vendor, each in duplicates). Bar diagrams, differential activation of analyte molecules upon Gα12 (A) or Gα13 (B) knockdown, presented as x-fold over control. C, Western blots to confirm the increased ERK1/2 activation upon Gα12 and/or Gα13 knockdown. Increased levels of pERK1 and pERK2 are observed in stable Gα12, Gα13, and double knockdown cells. D, increased proliferation of H69 cells upon treatment with the ERK inhibitor U0126.

Fig. 7.

Antibody arrays for the assessment of changes in the phosphorylation of various MAPK signal transduction pathways upon stable knockdown of Gα12 or Gα13. Arrays were probed with lysates from H69 cells stably transfected with control shRNA versus Gα12 shRNA (A) or with control shRNA versus Gα13 shRNA (B). Bottom, two representative examples of exposures per array used for quantitation (signals in the uppermost rows: loading controls; all other signals: analyte spots as dotted by the vendor, each in duplicates). Bar diagrams, differential activation of analyte molecules upon Gα12 (A) or Gα13 (B) knockdown, presented as x-fold over control. C, Western blots to confirm the increased ERK1/2 activation upon Gα12 and/or Gα13 knockdown. Increased levels of pERK1 and pERK2 are observed in stable Gα12, Gα13, and double knockdown cells. D, increased proliferation of H69 cells upon treatment with the ERK inhibitor U0126.

Close modal

For some signal transduction molecules, distinct differences between Gα12 and Gα13 knockdown were observed, supporting the notion that Gα12 and Gα13 may exert nonredundant functions in SCLC. For example, Gα12 knockdown resulted in a profound reduction of HSP-27, RSK1, RSK2, and p38δ activation (Fig. 7A), whereas signals in Gα13 cells (Fig. 7B) remained unchanged (RSK1, RSK2, and p38δ) or were strongly increased (HSP-27). The activation of other target proteins was only moderately changed upon Gα12 or Gα13 knockdown.

Most prominent, however, were the changes in ERK1/2 activation. Stable Gα12 knockdown led to a marked activation of ERK1 (>2.5-fold) and ERK2 (>2-fold; Fig. 7A). Similar results were obtained in stably Gα13 shRNA–expressing cells (Fig. 7B) indicating a tonic inhibition of the ERK pathway in SCLC by both Gα12 and Gα13. ERK1/2 activation was also seen in Western blots that showed a 1.5- to 2.7-fold increase of ERK2 phosphorylation upon stable knockdown of Gα12 and/or Gα13 (Fig. 7C), thus confirming the results from the antibody arrays. Because these data suggest that hyperactivity of ERK1/2 signaling could be responsible for the observed antiproliferative effects of Gα12 or Gα13 knockdown, it was also tested whether the inhibition of ERK1/2 could in turn induce proliferation. Indeed, the treatment of H69 cells with the ERK inhibitor U0126 led to increased proliferation (Fig. 7D).

The dependence of SCLC on mitogenic signaling of G proteins has been well established, and a mitogenic function of G12/13 signaling has been discussed because the expression of WT Gα12 is sufficient to confer malignant growth characteristics on fibroblasts (6, 7). However, the contributions of Gα12 and of Gα13 to the malignant phenotype of SCLC still remain elusive. Furthermore, the transforming activities of Gα12 and Gα13 were shown to be not redundant because overexpression of Gα13 does not exhibit effects in fibroblasts comparable with Gα12 (34). This requires a separate assessment of Gα12 and Gα13 functions in SCLC. In breast and prostate cancer cells, previous studies showed a role of Gα12 and of Gα13 in invasiveness, but not in tumor cell proliferation (9, 10).

In contrast, we show in this article that in SCLC cells, the downregulation of either Gα12 or Gα13 leads to a clear inhibition of proliferation in vitro as well as in vivo. Strikingly, the in vivo data also reveal an additive effect of Gα12 and Gα13 depletion, with double targeting of both proteins leading to complete abolishment of tumor growth. This seminal finding further supports the concept that intact G12/13 signaling is a prerequisite for tumor growth in SCLC.

Expression of shRNA against Gα12 or Gα13 by a lentiviral system elicited a sustained and efficient downregulation of the respective target in SCLC cells, an effect still detectable after 2 months in vitro culture or after >1 month in vivo growth in xenografted nude mice. This long-term stability reflects the genomic integration of lentivirally delivered shRNA-coding constructs, thus allowing the loss-of-function analyses done here. The stable downregulation used here, as opposed to transient targeting or stimulation approaches, also mimicks a prolonged therapeutic intervention. Furthermore, the use of shRNA-mediated gene targeting rather than inhibitors allowed to precisely distinguish between G12 and G13 signaling regarding the biological function. Notably, the persistent downregulation of Gα12 did not lead to a compensatory upregulation of Gα13 and vice versa. Likewise, no changes in Gα11 or Gαq expression were observed. The latter observation as well as the correlation between knockdown efficacies and antiproliferative effects of the various shRNA constructs also suggests the absence of off-target effects, although the possibility of nonspecific effects can never be completely excluded.

Regarding G protein–dependent signaling and its effects on proliferation of SCLC cells, the best characterized pathway emanates from the activation of Gq/11 (e.g., through stimulation with neuropeptides), which leads to the activation of the phospholipase Cβ-PKC cascade and to a increase in intracellular calcium ([Ca2+]i; ref. 21). Elevated [Ca2+]i stimulates ERK1/2 through the activation of Pyk2 and Src kinases, and subsequently Ras (35). In contrast, our knowledge on biological functions and molecular effectors of G12/13 signaling (11) in SCLC cells is rather limited. Neuropeptide-promoted stimulation of G12/13 has been shown to lead to the activation of monomeric GTPases of the Rho family (20, 36), and a pivotal role of Rho proteins as effectors of G12/13 signaling in SCLC cells is suggested by the fact that Rho proteins show particularly high expression levels in these tumors (23, 37). Rho and Rho kinase are involved in transendothelial migration of SCLC cells (38). Furthermore, a G12/13 and Rho-dependent signaling pathway has been described involving the activation of Pyk2, which could represent a point of convergence for Gq/11 (see above) and G12/13-dependent signaling (39). However, the inactivation of Rho proteins through clostridial C3 exoenzyme did not alter the proliferation of SCLC cells in our study, whereas proliferation of NSCLC cells was decreased (22, 23). This agrees with our findings that the pharmacologic inhibition of Rho kinase, a downstream effector of Rho proteins, did not inhibit the growth of H69 and H209 cells. Thus, we conclude that the activation of the Rho-Rho kinase pathway does not seem to represent a critical component of the G12/13-dependent proliferation pathways.

On the other hand, numerous G12/13-dependent and Rho-independent potential mitogenic pathways have been suggested, which are mediated, e.g., through Rac (40, 41), ERK5 (42), and the cadherin-catenin complex (43, 44). Thus, other molecular effectors are potential candidates to contribute to the G12/13-dependent mitogenic signaling in SCLC cells. In fact, our data obtained from the antibody array indicate that additional pathways that have not been implicated in G12/13 signaling thus far seem to be involved. Conversely, certain signal transduction modules previously described as downstream effectors of G12/13 do not seem to play a major role in SCLC. For example, it was shown in SCLC cells that G12/13 stimulation through UV radiation or treatment of cells with G12/13-stimulating biased agonists inhibits proliferation through the activation of c-Jun-NH2-kinase (24, 45). This conclusion is not supported by our experiments at least for the SCLC cell line used here because in the antibody array, only minor changes, if any at all, are observed upon stable Gα12 or Gα13 knockdown. However, it should also be noted that previous studies relied on short-term agonist treatment, whereas in our experiments, stable cell lines were analyzed. Thus, potential adaptive processes upon prolonged inhibition of G12/13 signaling have to be taken into account (see below).

12, but not Gα13, knockdown led to a marked >50% inactivation of Hsp27. Heat shock proteins including Hsp27, which are induced in cells exposed to stress and which function as molecular chaperons involved in protein folding, have been implicated in cancer (46), and Hsp27 is expressed in the majority of non-SCLC (47). Because Hsp27 is involved in cell growth, the reduction of activated Hsp27 upon G12 knockdown may add to the antiproliferative effects observed in our study. In contrast, stable targeting of Gα13 even results in the enhanced phosphorylation levels of Hsp27. This observation underlines that G12 and G13 signaling targets different downstream effectors and further strengthens the concept of nonredundant functions of G12 and G13 pathways.

The most pronounced effect in response to Gα12 or Gα13 knockdown was an increased ERK1/2 activation as detected by the antibody array and subsequently confirmed by Western blotting with phosphoERK1/2-specific antibodies. ERK1/2 activity usually correlates positively with cell proliferation—a notion that seems to contradict the cellular phenotype observed in our stably G12/13-downregulated cell lines. However, the finding of growth suppression upon hyperactivity of ERK1/2 was supported by our ERK inhibitor experiments, and a more complex function of ERK1/2 has emerged by detailed studies in different cell models. In fact, in the case of SCLC, it has been shown that overstimulation of the Ras-MAP/ERK kinase-ERK cascade following the expression of constitutively active Ras or the overexpression of Raf-1 leads to growth arrest and apoptosis (13, 4850). Thus, the elevated basal phosphorylation of ERK1/2 upon Gα12 or Gα13 knockdown may indicate an analogously disbalanced signaling. One possibility in this context would be that the inhibition of G12/13 signaling leads to the increased activation of Gq/11-dependent pathways. Gq/11-promoted signaling has been shown to stimulate the Ca2+-dependent tyrosine kinase Pyk2 and to subsequently cause the activation of the Ras-ERK cascade (35). In a balanced signaling context, this pathway exerts mitogenic effects in SCLC cells (35). In contrast, overexpression of Pyk2 in SCLC cells leads to growth arrest and apoptosis,3

3T. Büch, T. Gudermann, A. Aigner, unpublished data.

suggesting that the uncontrolled activation of this pathway is antiproliferative in SCLC cells. However, Gα12 or Gα13 knockdown affected neither basal Ca2+ levels (Supplementary Fig. S2) nor the bradykinin-stimulated increase in Ca2+ concentration (Supplementary Fig. S3). This suggests that the downregulation of Gα12 or Gα13 does not interfere with Gq/11-mediated signaling.

Depending on the cell type, ERK1/2 activation has been shown to occur through different pathways that distinctly define the duration and cellular distribution of activated ERK1/2. More specifically, RSK activation seems to be achieved only by transient rather than sustained activation of ERK1/2 (51). As the antibody array data obtained from unstimulated cells reflects constitutive activation or inactivation of signaling proteins, the parallel increased phosphorylation status of ERK1/2 and the concomitant dephosphorylation and inactivation of RSK in Gα12-depleted cells conforms to the latter concept.

Taken together, our data show that Gα12 and Gα13 knockdown results in multifacetted cellular responses that will require further analysis. The selective knockdown of Gα12 and Gα13 in SCLC cells presented herein proves that both proteins, Gα12 and Gα13, exert nonredundant proliferative effects in SCLC cells. It represents a promising approach to identify the downstream targets of these G proteins critically involved in the proliferation in SCLC cells and may help in further dissecting the nonredundant functions of Gα12 and Gα13 in these tumors. The complete abolishment of tumor growth upon parallel shRNA-mediated targeting of Gα12 and Gα13 also shows the increased therapeutic efficacy of RNAi-based double knockdown approaches of nonredundant, but closely related tumor-relevant gene products.

No potential conflicts of interest were disclosed.

We thank Johanna Platzek, Damian Stebel, and Andrea Wüstenhagen for the expert help with the experiments; Fatma Aktuna for preparing the tissue sections; and Didier Trono for providing pLVTHM, pMD2G, and psPAX2 plasmids.

Grant Support: Deutsche Forschungsgemeinschaft SFB-Transregio 17 (T. Gudermann and A. Aigner) and Deutsche Forschungsgemeinschaft-Forschergruppe 627 (A. Aigner).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data