1. INTRODUCTION

ABB

Advances in Bioscience and Biotechnology

2156-8456

Scientific Research Publishing

10.4236/abb.2014.51011

ABB-42364

Articles

Biomedical&Life Sciences

Cell-type specific and non-redundant anti-proliferative effects of shRNA-mediated Galpha12- and Galpha13 knockdown in lung cancer cell lines

homas

R. H. Büch

¹Marius

Grzelinski

²Olaf

Pinkenburg

³Thomas

Gudermann

⁴Achim

Aigner

⁵^*

Rudolf-Boehm-Institute for Pharmacology and Toxicology, Uni-versity of Leipzig, Leipzig, Germany; Biochemia Pharmacological Institute/bpc, Philipps-University Marburg, Marburg, Germany

Rudolf-Boehm-Institute for Pharmacology and Toxicology, Uni-versity of Leipzig, Leipzig, Germany; Biochemia Pharmacological Institute/bpc, Philipps-University Marburg, Marburg, Germany; Present Address: Institute for Immunology, Philipps-University Marburg, Marburg, German

Rudolf-Boehm-Institute for Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany; Biochemia Pharmacological Institute/bpc, Philipps-University Marburg, Marburg, Germany

Walther Straub Institute of Pharmacology and Toxicology, University of Munich (LMU), Munich, Germany; Comprehensive Pneumology Center Munich (CPC-M), German Center for Lung Research, Munich, Germany

* E-mail:achim.aigner@medizin.uni-leipzig.de(AA);

03012014

0501738013 November 201324 December 2013 13 January 2014

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G_12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G_12/13 signaling is less explored. While in prostate and breast cancer, G_12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G_12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G_12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα₁₂, Gα₁₃, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα₁₂ and Gα₁₃ knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα₁₂ or Gα₁₃ was sufficient to impair proliferation and double knockdown of Gα₁₂ and Gα₁₃ tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα₁₂ and Gα₁₃ affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.

KEYWORDS Galpha12; Galpha13; G<sub>12/13</sub>; Lung Cancer; SCLC; NSCLC; Gene Knockdown

1. INTRODUCTION

Lung cancer represents the leading cause of cancer-related death in the industrialized countries. Cancers of the lung are subdivided into different histological subtypes, e.g., adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma (SCLC). The former three subtypes are often subsumed as non-small cell carcinoma (NSCLC). Lung cancer of the SCLC type shows a very aggressive behavior with early manifestation of metastases. Thus, local therapeutic options like surgery are usually inapplicable in this tumor entity at the time of diagnosis. While current chemotherapy regimens in SCLC tumors clearly show clinical benefit, the overall survival after 5 years is still only 7% - 12% [1]. In NSCLC, although the potential to develop metastases is generally somewhat lower as compared to SCLC, dissemination is frequent at the time of diagnosis, necessitating systemic chemotherapy. This clearly emphasizes the need for novel treatment strategies based on the detailed understanding of molecular events underlying SCLC and NSCLC tumorigenesis and tumor progression.

In both SCLC and NSCLC cancer cells, growth and survival is critically regulated by a number of G proteindependent pathways. In SCLC cells, these signaling pathways are constitutively activated by the secretion of diverse autocrine stimuli, e.g., bradykinin, galanin, bombesin, or acetyl choline. The auto-stimulatory ligands activate G protein-coupled receptors (GPCR), which signal via the parallel activation of G_q/11 and G_12/13 proteins. Activation of G_q/11-dependent pathways has been shown to lead to the stimulation of the Ras-ERK1/2 cascade [2,3]. In contrast, the distal effector molecules of G_12/13 proteins in SCLC cells are less characterized. However, an important role of G_12/13 proteins in this tumor entity has been suggested by the fact that shRNA-mediated downregulation of Gα₁₂ or Gα₁₃ proteins in the SCLC cell lines H69 and H209 led to a marked growth inhibition [4].

In this study, we selectively disrupted G₁₂ and G₁₃ function by knockdown of Gα₁₂ or Gα₁₃, respectively, in order to decipher their individual relevance in different lung cancer cell lines. RNAi-mediated specific gene targeting of the α-subunits of G₁₂ or G₁₃ (Gα₁₂ or Gα₁₃), alone or in combination, was achieved by infection of the SCLC cell lines H510, H82 and the NSCLC cell lines A549 and H441 with shRNA constructs using a lentiviral system. While single targeting revealed, cell-specifically, a profound anti-proliferative effect in most cell lines in response to Gα₁₂ or Gα₁₃ downregulation, double targeting of both Gα₁₂ and Gα₁₃ did not yield any additive effects. Thus, we show that both Gα₁₂ and Gα₁₃ play a critical role in SCLC and NSCLC proliferation in vitro, and that selectively interfering with Gα₁₂ and Gα₁₃ signaling, e.g. through RNAi-mediated therapeutic blockage of both G proteins, may provide a promising new avenue in lung cancer treatment.

2. MATERIALS AND METHODS2.1. Viral shRNA Construct, Cell Lines, and Stable Transfection

Specific shRNA expression plasmids were generated as described previously [4], Briefly, chemically synthesized (MWG Biotech) oligonucleotides designed according to [5,6] were cloned into the pSUPER vector using the BglII and HindIII restriction sites, and correct insertion was controlled by sequencing. Using ClaI and BamHI, the H1-cassettes of the pSUPER vectors were then cloned into the lentivirus vector pLVTHM. For recombinant virus generation, 20 μg/10 cm dish of the cis-vectors were co-transfected into HEK cells together with 15 μg psPAX2 (“trans plasmid”) and with 6 μg pMD2G (VSV-G) using the calcium phosphate precipitation method. 6 - 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, upon resuspension in complete RPMI medium, the viral titer was determined by serial dilution in HEK cells.

Lung cancer cell lines H82, H510, A549, and H441 were obtained from the American type culture collection (ATCC) and cultivated under standard conditions (37˚C, 5% CO₂) in RPMI medium (PAA, Cölbe, Germany) supplemented with 10% fetal calf serum (FCS). H82, H510, A549, and H441 cells were transduced with 20 TU (transducing units) of recombinant virus, and after 48 h selected for stable integration by supplementing the medium with G418.

2.2. RNA Preparation and Quantitative RT-PCR

Total RNA from tumor cells was isolated using the Tri reagent (PEQLAB, Erlangen, Germany) according to the manufacturer’s protocol. Reverse transcription was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit from Fermentas (St. Leon-Rot, Germany) as follows: 2 μg total RNA and 1 μl random hexamer primer (0.2 μg/μl) were diluted in DEPC-treated water ad 11 μl, incubated at 70˚C for 5 min and chilled on ice prior to adding 4 μl 5X reaction buffer, 0.5 μl RNAse inhibitor (20 u/μl), 2 μl 10 mM dNTP 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, prior to stopping the reaction by heating at 70˚C for 10 min and chilling on ice. Quantitative PCR was performed in a Light Cycler from Roche (Penzberg, Germany) 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 μM each) and 5 μl SYBR Green master mix. A pre-incubation for 15 min at 95˚C was followed by 55 amplification cycles: 10 sec at 95˚C, 10 sec at 55˚C and 10 sec 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 sec prior to heating to 95˚C. To normalize for equal mRNA/ cDNA amounts, PCR reactions 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 ΔΔC_t method with β-actin as reference housekeeping gene.

2.3. Proliferation Assays

Cell lines were plated into 96 well plates at 2000 - 4000 cells/well and 5 wells/time point and cultivated in RPMI/ 10% FCS in a humidified incubator under standard conditions. At the time points indicated, the number of viable cells was determined using a colorimetric assay. Briefly, the medium was aspirated and 50 µl of a 1:10 dilution of Cell proliferation Reagent WST-1 (Roche Molecular Biochemicals, Mannheim, Germany) in medium was added to the cells prior to incubation for 1 h at 37˚C. The absorbance at 450 nm was measured using a Dynex MRX microplate reader (Pegasus Scientific Inc., Rockville, MD).

3. RESULTS3.1. Stable Expression of shRNAs Leads to Specific Knockdown of Gα12 or Gα13

Using a lentiviral expression system, the expression of shRNAs directed against Gα₁₂ or Gα₁₃ in H510 SCLC cells led to a stable 70% (Gα₁₃) to 80% (Gα₁₂) downregulation of the respective target mRNA as compared to negative control-transfected (“unrelated”) H510 cells (Figures 1(a) and (b)). In this cell line, the double transfection with simultaneous expression of both Gα₁₂- and Gα₁₃-specific shRNAs resulted in a lower downregulation efficiency of Gα₁₂ mRNA as compared to cells expressing only Gα₁₂-specific shRNA (Figure 1(a)). Importantly, in single-transfected cells expressing either Gα₁₂ or Gα₁₃-directed shRNAs, no counter upregulation of the other G protein was discernible (Figures 1(a) and (b)). This finding suggests that an upregulation of Gα₁₂ cannot compensate for the knockdown of Gα₁₃ and vice versa, thus indicating a non-redundancy in the function of both proteins. This was further explored in proliferation assays.

3.2. Anti-Proliferative Effects Differ between Gα12 or Gα13 Knockdown and Are Cell Line Specific

Regarding the effects of Gα₁₂- and Gα₁₃ knockdown on proliferation in H510 cells, a ~50% inhibitory effect was found after single transfection with Gα₁₃ shRNA. The double transfection with Gα₁₂ and Gα₁₃ shRNAs yielded similar rates of growth inhibition (Figure 1(c)). The most pronounced effect, however, was observed upon Gα₁₂ single knockdown with a >90% reduction in tumor cell proliferation, suggesting a more dominant role of Gα₁₂ in this cell model. The difference between Gα₁₂ single vs. Gα₁₂ + Gα₁₃ double knockdown also indicates that the degree of Gα₁₂ knockdown is critical, since the double infection had yielded higher residual Gα₁₂ levels as compared to the single knockdown.

To further evaluate the role of Gα₁₂ or Gα₁₃ signalling in SCLC cells, we tested another SCLC cell line. H82 belongs to the variant type of SCLC cells as opposed to H510 cells which represent a classic type SCLC cell

line. As in H510 cells, the expression of Gα₁₂ or Gα₁₃- specific shRNAs led to a specific at least 60% downregulation of the target G protein without affecting the non-targeted G protein (Figures 2(a) and (b)). Again, no compensatory (up-) regulation of the non-targeted G protein was observed, thus confirming the previous finding in H510 cells.

However, in contrast to H510 cells and despite the efficient knockdown, the anti-proliferative effect after shRNA expression was generally lower and showed a different pattern regarding the contributions of the individual G proteins. In fact, the strongest anti-proliferative effects occurred after Gα₁₃ inhibition, either in the single or the double knockdown setting, whereas Gα₁₂ inhibition (which had the strongest impact in H510 cells; see above) did not induce growth inhibition when compared to cells expressing unrelated shRNA (Figure 2(c)). Thus, the effects of interference with Gα₁₂ or Gα₁₃ appear to be cell-type specific.

3.3. Inhibition of Gα12 or Gα13 Expression in the NSCLC Cell Lines

Extending our studies towards NSCLC, we tested the role of Gα₁₂ and Gα₁₃ in A549 cells. This cell line shows characteristics of adenocarcinoma cells and resembles type II alveolar cells. In these cells, the efficiency of shRNA-mediated knockdown of the Gα subunits was overall lower (albeit still significant) as compared to H510 or H82 cells, with a ~50% (Gα₁₂) to ~60% (Gα₁₃) residual levels (Figures 3(a) and (b)). Again, the double knockdown yielded somewhat less Gα₁₂ knockdown efficacy as compared to the single targeting of Gα₁₂. In this cell line, despite profound overall proliferation, no antiproliferative effect of the shRNAs was discernible (Figure 3(c)). Even the parallel Gα₁₂ and Gα₁₃ double knockdown did not show a trend towards growth inhibition. One possibility is that cell-type specific differences were responsible for the missing shRNA effects. Alternatively, this lack of anti-proliferative effects could be potentially attributable to the lower knockdown efficiency in these cells as compared to the SCLC cell lines. To further test these assumptions and to further analyse if Gα₁₂ or cells Gα₁₃ knockdown can lead to antiproliferation in NSCLC cells, another NSCLC cell line with a different biological background, H441, was employed.

This cell line has features of adenocarcinoma cells but, in contrast to A549 cells, does not resemble alveolar type II cells while rather showing differentiation markers of club cells. Comparable to A549 cells, the efficiency of shRNA-mediated downregulation of Gα₁₂ and Gα₁₃ in H441 was generally lower when compared to the SCLC cell lines (Figures 4(a) and (b)). Despite the only 30% - 35% knockdown of Gα₁₂ and Gα₁₃, however, marked ~50% anti-proliferative effects were observed (Figure 4(c)). Thus, the relatively lower efficiency of shRNAs against Gα₁₂ and Gα₁₃ does not account (alone) for the lack of effects on proliferation in A549 cells. Rather, cell inhibi-

tory effects upon Gα₁₂ or Gα₁₃ gene targeting appear to be cell line specific, with at least in some cases rather

mild knockdowns being sufficient. In line with this, the individual contributions of Gα₁₂ and Gα₁₃ also seem to depend on the cell line, since in H441 cells a largely identical growth inhibition upon Gα₁₂ or Gα₁₃ single knockdown was observed, unlike in H510 (Gα₁₂ effects more profound) or H82 cells (Gα₁₃ knockdown more important).

4. DISCUSSION

Heterotrimeric G proteins are critically involved in the regulation of virtually every physiologic and pathophysiologic process. Since G protein-dependent signaling plays a pivotal role in the regulation of cellular growth, survival, motility, and differentiation, it does not come as a surprise that these proteins are important signaling molecules in cancer formation and progression and thus represent promising drug targets [7-10]. According to the different α subunits, these proteins are subdivided into the G_s, G_i/o, G_q/11, and G_12/13 subfamily [11].

Of note, one of the first identified biological functions of the G_12/13 subfamily was its propensity for neoplastic transformation, as shown after overexpression of wildtype Gα₁₂ in mouse fibroblasts [12]. Moreover, in a number of studies a marked oncogenic potential of the Gα₁₂ and Gα₁₃ subunits has been described upon overexpression of GTPase deficient (and therefore constitutively active) variants [13,14]. Additionally, it has been shown that the stimulation of G_12/13-coupled receptors can also induce neoplastic transformation [15,16]. Thus, even in the presence of endogenous expression levels of wildtype G_12/13 proteins, the functional overactivation of these signaling molecules can contribute to a malignant phenotype. However, the tumorigenic effects of G_12/13-dependent signaling may well be dependent on the tumor entity and cell-type [17]. Thus, the role of the individual protein (Gα₁₂ or Gα₁₃) has to be elucidated separately, i.e. apart from the other in every tumor entity, and many studies so far have relied on the somewhat non-natural ectopic overexpression of Gα₁₂ or Gα₁₃. The latter requirements make the RNAi-mediated knockdown a particularly well-suited approach for more detailed functional analyses.

One problem in the understanding of tumor signaling via G_12/13 proteins is that a multitude of downstream effector molecules exist (see [18,19] for review). For this reason, the mechanistic basis of oncogenic signaling of G_12/13 proteins is still widely unsettled. Besides a classic pathway that is regulated by G_12/13-dependent cascades and affects the actin cytoskeleton via stimulation of Rho family proteins [20], thus leading to alterations in cell motility and consequently in their metastatic and invasive potential [21], a role of G_12/13 proteins in proliferation or cellular survival has been emphasized by a number of studies [4,22]. Indeed, with regard to lung cancer cells, we demonstrated previously that the knockdown of Gα₁₂ or Gα₁₃ severely impairs the proliferation in the SCLC cell lines H69 and H209 [4]. This is in line with reports on the relevance of G_12/13-coupled neuropeptide receptors [23] and on the constitutive secretion of autostimulatory agonists for G_12/13-coupled receptors (for review: [2]).

Given the aforementioned cell-type specific roles of G_12/13 signaling, however, it was critical to determine whether these previous findings were also attributable to other SCLC cell lines and could even be extended towards NSCLC. Indeed, the results presented herein suggest that an important role of G_12/13 proteins in the regulation of proliferation can be also found in NSCLC cells like H441. However, the findings in the NSCLC cell line A549, where inhibition of G_12/13 did not produce a growth inhibiting effect, underpin the importance of the cellular context for G_12/13-dependent mitogenic effects.

Since G₁₂ and G₁₃ signaling affects overlapping target molecules, potentially redundant functions of these two G proteins are an important issue. Besides parallel, overlapping activities, this also includes the question if an enhancement in Gα₁₂ signaling could compensate for the inhibition of Gα₁₃ in tumor cells and vice versa. If so, the therapeutic efficacy of any intervention targeting only one of the two G proteins would be greatly diminished. The highly specific, RNAi-based knockdown in stable cell lines provides an excellent model to test the presence or absence of counter-regulatory effects. Importantly, the shRNA-mediated downregulation of Gα₁₂ or Gα₁₃ mRNA did not produce an upregulation of the non-targeted G protein. Moreover, despite the in different degrees of functional relevance of both G proteins in different cancer cells as outlined above, this was true in any of the investigated cell lines. It should also be noted that, unlike seen in other oncogene families (see e.g. [24] for HER receptor), the double targeting of both Gα₁₂ and Gα₁₃ did not lead to synergistic anti-proliferative effects. In tumor cells insensitive to a single knockdown of Gα₁₂ or Gα₁₃ (see A549 experiments), the double targeting of both G proteins had no anti-proliferative effect either. This demonstrates that the absence of a cellular dependence on Gα₁₂ or Gα₁₃ cannot simply be explained by the compensatory signaling through the other, and implies that other cellular or molecular features determine the cellular dependence on Gα₁₂ and Gα₁₃. More importantly from a possible therapeutic viewpoint, in cells sensitive to inhibition of G_12/13 signaling like H510, H82, and H441, the targeting of one of the two G proteins is sufficient to impair proliferation.

Finally, one has to bear in mind that many G proteincoupled receptors constitutively activated in tumor cells propagate signaling by activating G_12/13 and G_q/11 proteins in parallel [25,26]. Therefore, the downregulation of Gα₁₂ or Gα₁₃ may lead to a relative overactivation of G_q/11-dependent pathways. Thus, dysbalanced G protein signaling rather than the parallel activation of G_12/13 and G_q/11 proteins may lead to an inhibition of proliferation, and may thus contribute to the antitumor effects and their cell line-specificity observed here upon downregulation of Gα₁₂ or Gα₁₃.

DISCLOSURE STATEMENT

The authors declare no competing financial interests.

ACKNOWLEDGEMENTS

We acknowledge the skillful technical assistance of Bärbel Obst. The project was funded by a grant from the Deutsche Forschungsgemeinschaft (DFG; Gu265/6-1, Ai 24/10-1) and by the German Center for Lung Research and the BMBF (German Ministry of Education and Research).

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