hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing (original) (raw)
The alternative splicing of caspase-9 is dysregulated in NSCLC tumors and cell lines. In this study, the ratio of caspase-9a/9b mRNA was compared between human lung tumor samples and normal human lung tissue (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI43552DS1). Tumor samples were subdivided into 3 groups: normal, with a caspase-9a/9b mRNA ratio of at least 3.5; moderately dysregulated, with a 2.3–3.4 ratio of caspase-9a/9b mRNA; and highly dysregulated, with a caspase-9a/9b mRNA ratio of 2.2 or less (Figure 1, A and B). The normal group corresponded to the normal ratio of caspase-9a/9b mRNA observed in non-transformed cells; the moderately dysregulated group corresponded to a ratio of caspase-9a/9b reported to have a minor to moderate effect on caspase-9 activity (5, 6); and the highly dysregulated group corresponded to a ratio of caspase-9a/9b reported to significantly reduce caspase-9 activity and inhibit the association of caspase-9a with APAF-1 (5, 6). Quantitative/competitive RT-PCR analysis (16) of caspase-9 splice variants (17) showed that 40% of the NSCLC tumors examined (n = 81) presented with a highly dysregulated ratio of caspase-9a/9b mRNA compared with normal lung tissue. Only 15% of the lung tumor samples demonstrated a normal ratio of caspase-9a/9b mRNA, and 45% of the lung tumor samples demonstrated a moderately dysregulated ratio of caspase-9a/9b mRNA. The entirety of the human lung tumors were also compared directly with normal lung tissue, and demonstrated a reduction of 36% in the caspase-9a/9b mRNA ratio in human lung tumors (Figure 1C). Thus, the ratio of caspase-9a/9b mRNA was dramatically lower in a high percentage of clinically relevant NSCLC tumors, correlating with loss of activity of caspase-9a and favoring a pro-survival/apoptotic resistance phenotype.
Caspase-9a/9b ratio is dysregulated in lung adenocarcinoma tumors and transformed lung epithelial cells. (A) Top: A sample population of cDNAs from pathologist-verified lung adenocarcinomas, which underwent quantitative/competitive PCR for caspase-9 splice variants. Bottom: The figure depicts a sample of the matched pair analysis. N, normal tissue; T, tumor tissue. (B) The percentage of lung tumors grouped by caspase-9a/9b mRNA ratio. (C) Human lung tumor samples directly compared with normal lung tissues. *P < 0.05, Student’s t test. (D and E) The caspase-9 splicing ratio observed in non-transformed HBEC-3KT cells versus NSCLC cell lines at the RNA (D) and protein level (E). Data are n = 3 on 2 separate occasions. (F and G) Top: Quantitative/competitive RT-PCR for caspase-9 splice variants from A549 cells treated with E4 ASRO (E4) or control (Con) ASRO (F) or control siRNA or siRNA against caspase-9b (9b-si) (G). Protein samples from simultaneous experiments were subjected to SDS-PAGE and Western immunoblotted (caspase-9b, α-tubulin). Bottom: Caspase-9 activity (LEHD) in the lysates of A549 cells treated with control ASRO and E4 ASRO. Caspase-9 activity (LEHD) was calculated as relative fluorescent units (RFU) per total protein (mg) and is represented as a percentage of control cells. The data presented in F and G are expressed as mean ± SEM and are n = 4 on 3 separate occasions.
Although all of our samples were more than 50% NSCLC tumors, contamination of other cell types may have affected results. Therefore, we examined a pure population of non-transformed lung epithelial cells (specifically, HBEC-3KT cells) for their caspase-9a/9b ratio in comparison with the transformed lung adenocarcinoma cell lines A549, H838, and H2030 (see Supplemental Table 2 for cell line phenotypes). HBEC-3KT cells demonstrated a “normal” ratio of caspase-9a/9b mRNA of 4.5 ± 0.08 (Figure 1D). In comparison to the non-transformed cell line, all 3 adenocarcinoma cells lines (A549, H838, and H2030) demonstrated a dysregulated ratio of caspase-9a/9b mRNA: A549, 2.2 ± 0.14; H838, 1.4 ± 0.08; H2030, 2.6 ± 0.12 (Figure 1D). Importantly, the dysregulation in the ratio of caspase-9a/9b mRNA translated to the protein level (Figure 1E). These observations indicate that the alternative splicing of caspase-9 is dysregulated in lung adenocarcinoma cell lines as well as in NSCLC tumors.
To verify that the observed differences in caspase-9a/9b mRNA ratio translated to a significant effect on caspase-9 activity, siRNA and anti-sense RNA oligonucleotide (ASRO) technology was employed to directly manipulate the alternative splicing of caspase-9. Specifically, ASROs were designed to hybridize to the 5′ splice site (5′SS) of exon 3 (E3 ASRO), exon 4 (E4 ASRO), and exon 6 (E6 ASRO) in caspase-9 pre-mRNA to reduce the ratio of caspase-9a/9b mRNA as previously reported for other splicing events (18–20). The E4 ASRO effectively reduced the ratio of caspase-9a/9b mRNA in cells (Figure 1F), which translated to the protein level in comparison with control ASRO. ASROs targeting the 5′SS of exon 3 and exon 6 also significantly inhibited the inclusion of the exon 3,4,5,6 cassette into the mature mRNA, but the E4 ASRO was the most effective ASRO (data not shown). These results suggest that all exons within the exonic cassette require appropriate definition for caspase-9a mRNA to be produced, or the default exclusion of the exonic cassette, and subsequent production of caspase-9b, will occur.
To examine the functional translation of directly manipulating the alternative splicing of caspase-9, total caspase-9 activity was examined with E4 ASRO in comparison with the control ASRO. Figure 1F demonstrates that the total caspase-9 activity (LEHD activity) was lowered approximately 62% by the E4 ASRO, corresponding to a 46% reduction in the caspase-9a/9b mRNA ratio as compared with the control ASRO. Thus, the E4 ASRO directly manipulated the alternative splicing of caspase-9 to produce caspase-9b at the expense of caspase-9a, which significantly lowered caspase-9 activity in the cell. In order to specifically reduce caspase-9b levels, RNAi technology was employed to downregulate caspase-9b (siRNA against caspase-9b [9b-si]) versus caspase-9a. Treatment with 9b-si in comparison with control siRNA significantly increased the ratio of caspase-9a/9b mRNA in A549 cells producing a caspase-9a/9b mRNA ratio comparable to HBEC-3KT cells, which was conferred to the protein level (Figure 1G). Lastly, caspase-9 activity was assessed in A549 cells after 9b-si transfection in comparison with control siRNA, which demonstrated an approximate 2.7-fold increase in the caspase-9 activity (LEHD activity) (Figure 1G). The culmination of these data demonstrate that our observed changes in the caspase-9a/9b mRNA ratio in NSCLC cells and tumors correlate with a significant effect on the activity of caspase-9 and are thus physiologically relevant.
The alternative splicing of caspase-9 regulates the anchorage-independent growth and tumorigenic capacity of NSCLC cells. Based on the above findings and the knowledge that caspase-9a is a possible tumor suppressor (10), although controversial (21), we hypothesized that the alternative splicing of caspase-9 has a major role in maintaining the transformed phenotype of NSCLC cells. To test this hypothesis, multiple clones, and batch cultures of A549 cells stably expressing either caspase-9b cDNA or caspase-9b shRNA were produced (Figure 2A and Supplemental Table 3). Stable expression of caspase-9b shRNA essentially abolished the ability of A549 cells to grow in soft agar and reduced the mean diameter of cellular colonies (Figure 2, B and C, and Supplemental Figure 1). In contrast, low ectopic expression of caspase-9b significantly increased the ability of A549 cells to grow in soft agar (Figure 2, B and C). These effects did not require stable expression and did not result from integration artifacts, as short-term expression of caspase-9b shRNA and caspase-9b cDNA or use of stable batch cell lines produced comparable results (data not shown).
Caspase-9 regulates the tumorigenic capacity in A549 cells. (A) Total RNA was isolated from the listed clonal cell lines and analyzed by competitive/quantitative RT-PCR for caspase-9 splice variants. (B) Cells (2 × 103) were plated into 6-well dishes in soft agar and cultured for 14 days before the colony count. (C) Quantization (mean) of the number of colonies for the indicated clonal cell lines. n = 6. Data are mean ± SEM. *P < 0.005, A549 + shRNA control versus A549 + C9b shRNA; **P < 0.001, A549 + vector control versus A549 + C9b ectopic; Student’s t test. (D) Cell lines in A were injected into SCID mice (5 × 106), and tumor volume was measured at the end of 28 days as represented as average size of tumor (cm3). Data are mean ± SEM. *P < 0.05, A549 + control shRNA versus A549 + C9b shRNA; ***P < 0.005, A549 + vector control versus A549 + C9b ectopic Student’s t test. (E) H&E stain of the tumors presented in D with magnification ranging from ×4 to ×40.
These observations translated to other NSCLC cell lines, as stable expression of caspase-9b shRNA in H2030 and H838 cell lines also induced a dramatic loss in the ability of these cells to grow in soft agar (Supplemental Figure 2). In contrast, low ectopic expression of caspase-9b demonstrated a marked increase in the ability of H838 and H2030 cells to grow in soft agar (Supplemental Figure 2). Furthermore, the clonogenic capacity in the formation of stable expression clones of caspase-9 shRNA was decreased by a factor of 10 compared with control shRNA expression using the same viral MOI (data not shown).
The tumorigenic capacity of these A549 cell lines was also characterized using human A549 xenograph tumor formation in SCID mice. Figure 2D shows that stable downregulation of caspase-9b by shRNA technology also induced a complete loss of tumor formation in 10 of 12 injections. The remaining tumors that formed in the caspase-9b shRNA–expressing cells demonstrated a dramatic loss in tumor volume (Figure 2D). Pathological analysis confirmed the complete loss of tumor formation for the caspase-9b shRNA experiments. In contrast, low ectopic expression of caspase-9b induced a large increase in tumor volume (Figure 2D). Interestingly, a different pathological phenotype was observed in the 2 minor tumors formed from cell lines expressing caspase-9b shRNA (Figure 2E). Specifically, caspase-9b shRNA xenographs demonstrated a greater quantity of matrix compared with the A549 vector control xenographs and absence of gland-like structures and invasion into the adjacent tissue. Furthermore, the caspase-9b shRNA tumors formed from the caspase-9b shRNA cells lost major architectural features of adenocarcinomas, and an inflammatory response was also observed. Large quantities of neutrophils were also present, along with visible apoptotic bodies, but there was a distinct absence of necrosis. Lastly, the effect of the downregulation of caspase-9b on AIG and tumorigenic capacity was not due to the enhancement of spontaneous apoptosis rates, as no significant effect on these rates in the A549 caspase-9b shRNA cells was observed compared with vector control cells (data not shown). Overall, these data demonstrate that the loss of caspase-9b dramatically alters the tumorigenic capacity of NSCLC cells.
Identification of a purine-rich exonic splicing silencer in exon 3 of caspase-9 pre-mRNA. Given the biological findings, our laboratory examined the mechanistic regulation of the pre-mRNA processing of caspase-9. In an effort to identify exonic splicing enhancers (ESEs) and ESSs, our laboratory constructed a functional minigene to assay the inclusion or exclusion of the exon 3,4,5,6 cassette of caspase-9 in cells. This was accomplished by ligation of 3 DNA fragments of the caspase-9 gene into pcDNA 3.1–. The 3 fragments included a 603-bp fragment containing part of exon 2 and intron 2; a 3,962-bp fragment containing part of intron II, all of exons 3,4,5,6 and their intervening intronic sequences (all of the sequence for introns 3, 4, and 5), and part of intron 6; and a 431-bp DNA fragment containing part of intron 6 and part of exon 7 (Figure 3A).
Identification of an exonic splicing silencer in exon 3 of caspase-9 pre-mRNA. (A) Schematic of the functional minigene construct for caspase-9 constructed in pcDNA 3.1– zeocin with arrows indicating the location of primers used in the RT-PCR assay. (B) Schematic representation of the exonic splicing silencer (C9/E3-ESS) located in exon 3 of the caspase-9 gene. Asterisk indicates the location of the C9/E3-ESS purine-rich sequence. This figure also depicts the wild-type and mutagenic sequence of the C9/E3-ESS utilized in C and D. (C and D) A549, H838, H2030, and HBEC-3KT cells were transfected with the pcDNA 3.1– zeocin plasmid containing the caspase-9 minigene (C9 WT MG) (1 μg), exon 3 mutant minigene (E3 Mut MG) (1 μg), or empty vector control (1 μg) for 24 hours. Total RNA was extracted and analyzed by competitive/quantitative RT-PCR for the ratio of minigene caspase-9a/9b mRNA. Data are n = 4 from 2 separate occasions.
Within the exon 3,4,5,6 cassette, 10 purine-rich sequences were identified as possible ESEs, intronic splicing enhancers, ESSs, or intronic splicing silencers by (a) screening the exon 3,4,5,6 cassette for known RNA _cis_-elements (e.g., purine-rich regions), (b) using ESE finder 2.0 (22), and (c) using Splicing Rainbow (23) (Supplemental Figure 3). Replacement mutagenesis of these sequences identified a particular purine-rich sequence in exon 3 (Figure 3B and Supplemental Figure 3) that acted as an ESS. Mutation of this purine-rich sequence in exon 3, AGGGGA, resulted in a significant increase of the caspase-9a/9b mRNA ratio in A549 cells from 2.7 ± 0.05 to 5.3 ± 0.05 (P < 0.0002) (Figure 3C). Of the 10 purine-rich sequences mutated in the caspase-9 minigene, only the mutagenesis of this particular sequence had the effect of increasing the ratio of caspase-9a/9b mRNA. Mutation of this ESS also induced an increase in the ratio of caspase-9a/9b minigene mRNA in H2030 and H838 cells, but interestingly no effect on the C9a/C9b ratio was observed in non-transformed HBEC3-KT cells (Figure 3D). Therefore, the purine-rich sequence, AGGGGA, in exon 3 of caspase-9 pre-mRNA acts as an ESS for the inclusion or exclusion of the exon 3,4,5,6 cassette in transformed NSCLC cells and is referred to throughout as caspase-9 exon 3 ESS (C9/E3-ESS).
hnRNP L binds specifically to the exonic splicing silencer in exon 3 of caspase-9 pre-mRNA. To study the RNA _trans_-factors interacting with C9/E3-ESS, a fluorescein-tagged RNA oligonucleotide (RO) was produced corresponding to the purine-rich regions of exon 3, FL-5′ GAGAGUUUGAGGGGAAAU. EMSAs coupled with competitor studies demonstrated 3 specific proteins, RNA complexes associated with this sequence designated complex I, II, and III (Figure 4A). To identify the RNA _trans_-factors bound to the C9/E3-ESS, nanospray LC-MS/MS analysis was employed on the 3 complexes. Besides the normal contaminants such as keratin and HSP70, 2 RNA _trans_-factors were identified in the 3 RNA/protein complexes, both with x-corr values over 20. Specifically, hnRNP A2/B1 (36 kDa) was identified in complexes I and III, while hnRNP L (65 kDa) was identified in complexes II and III (Figure 4A).
hnRNP L binds specifically to the exon splicing silencer in exon 3 of caspase-9. (A) A 5′ FITC-tagged RO corresponding to the purine-rich sequences in exon 3 of caspase-9 was incubated in the presence of nuclear extract from A549 cells or IgG (control) and subjected to EMSAs. NSC1 or SC ROs were also added (100-fold molar excess) as indicated. Arrows indicate the 3 specific RNA-protein complexes. The RNA-protein complexes (complexes I, II, and III) were also subjected to nano-LC-MS/MS analysis. The RNA _trans_-factors depicted obtained x-corr values greater than 20. (B) A 5′ biotinylated wild-type C9/E3-ESS RO (Bio-C9/E3-ESS), 5′ biotinylated mutant ROs (Bio-Mut C9/E3-ESS), or a 5′ biotinylated nonspecific RO (Bio-NSC2) were incubated in the presence of nuclear extract from A549 cells or IgG (control), subjected to SDS-PAGE and Western immunoblotting analysis (anti-hnRNP L antibody; anti-hnRNP A2/B1 antibody). Unlabeled nonspecific ROs (e.g., NSC1) at a 100-fold molar excess were also added to the reactions as indicated. The corresponding supernatant from the Bio-WT C9/E3-ESS (Sup) shows the remaining RNA _trans_-factor after affinity purification. (C and D) The experiments in B were repeated, but with the addition (100-fold molar excess) of either unlabeled NSC1, unlabeled competitor ROs (RO1–RO4) or unlabeled SC, as indicated. Data in Figure 4 are n = 4 on 2 separate occasions. (E) A cartoon schematic indicating the locations of the hnRNP L and hnRNP A2/B1 interactions with exon 3 of caspase-9 pre-mRNA.
The specific interaction of these RNA _trans_-factors with exon 3 was verified by performing a biotin/streptavidin affinity assay using A549 nuclear extracts. Biotinylated ROs corresponding to the purine-rich regions of exon 3 were utilized for this affinity assay (Table 1): biotin-5′ GAGAGUUUGAGGGGAAAU (Bio-WT C9/E3-ESS), a mutant RO for the C9/E3-ESS; biotin-5′ GAGAGUUUGCTACTAAAU (Bio-C9/E3-ESS-mut); and a biotin-5′ non-specific control sequence (Bio-NSC2). Analysis verified that RNA _trans_-factors hnRNP L and A2/B1 specifically interacted with the purine-rich regions in exon 3 of caspase-9 (Figure 4B), as the non-specific RNA sequence (Bio-NSC2) did not show any interaction with these RNA _trans_-factors. More importantly, the Bio-C9/E3-ESS-mut, which possesses the same mutations utilized in the minigene assays in Figure 3, did not significantly interact with hnRNP L, in contrast to hnRNP A2/B1. Thus, both hnRNP L and hnRNP A2/B1 specifically interact with the purine-rich regions exon 3 of caspase-9 pre-mRNA, but only hnRNP L interacts specifically with the C9/E3-ESS sequence.
Designed ROs used in affinity-based assays
To determine where hnRNP A2/B1 specifically bound in exon 3 (e.g., AGGGGA) as well as identify other important nucleic acid residues for the hnRNP L interaction, ROs were designed that possessed mutations in various sites within the purine-rich regions of exon 3 (Table 2). In particular, RO4 and RO2 possessed similar and flanking mutations compared with the “core” mutations utilized to identify C9/E3-ESS using the caspase-9 minigene shown in Figure 3. The competition affinity-based assays used to obtain the results shown in Figure 4C were repeated using each mutant RO as an unlabeled “cold” competitor, in comparison with cold nonspecific competitor (NSC) and specific competitor (SC). RO4 was unable to compete for hnRNP L interaction with Bio-C9/E3-ESS (Figure 4C). Furthermore, RO2 was only a partial competitor for the hnRNP L/Bio-C9/E3-ESS interaction. In contrast, the hnRNP A2/B1 interaction with exon 3 was abolished to the same extent by RO4 and RO2 as an unmutated exon 3 SC. In further contrast, RO3 and to an extent RO1 were unable to compete for hnRNP A2/B1 binding in contrast to hnRNP L (Figure 4D). Therefore, hnRNP L specifically associates with C9/E3-ESS in exon 3 as well as flanking nucleotide sequences, whereas hnRNP A2/B1 associates with a purine-rich region 6–11 bp upstream of the previously identified C9/E3-ESS sequence (Figure 4E).
Designed ROs used in competition affinity-based assays
hnRNP L regulates the pre-mRNA processing of caspase-9 in NSCLC cells. Although hnRNP A2/B1 did not bind specifically to the C9/E3-ESS, compared with hnRNP L, a role for this RNA _trans_-factor in repressing the inclusion of the exon 3,4,5,6 cassette was still a possibility. siRNA technology was used to examine the role of both hnRNP A2/B1 and hnRNP L in regulating the ratio of caspase-9 splice variants. Downregulation of hnRNP L using a multiplex siRNA (100 nM [25 nM for each sequence]) resulted in a significant increase in the ratio of caspase-9a/9b mRNA from 2.2 ± 0.07 to 3.9 ± 0.07 (P < 0.005; n = 4) (Figure 5A). On the other hand, multiplex siRNA treatment against hnRNP A2/B1 had no significant effect on the ratio of caspase-9a/9b mRNA (Figure 5B). Western immunoblot analysis confirmed a more than 80% downregulation of both hnRNP A2/B1 and hnRNP L compared with samples from cells treated with control siRNA (Figure 5, A and B). Importantly, the effect of hnRNP L siRNA on the caspase-9a/9b mRNA ratio translated to the protein level (Figure 5C).
Downregulation of hnRNP L, but not hnRNP A2/B1, increases the ratio of caspase-9a/9b mRNA. A549 cells were transfected with control siRNA (100 nM), hnRNP L SMARTpool siRNA (100 nM), or hnRNP A2/B1 SMARTpool siRNA (100 nM) for 48 hours. Total RNA was isolated and analyzed by competitive/quantitative RT-PCR for caspase-9 splice variants. (A and B) hnRNP L siRNA (A) and hnRNP A2/B1 siRNA (B). Simultaneously, total protein lysates were also produced, subjected to SDS-PAGE analysis, and immunoblotted for hnRNP L, hnRNP A2/B1, caspase-9, and β-actin. (C) Results from the same Western blot membrane depicted in A. Data are n = 4 on 2 separate occasions.
The observed effect of hnRNP L downregulation on the caspase-9a/9b mRNA ratio could not be attributed to off-target effects, as multiple siRNA sequences targeted against hnRNP L produced the same result (Supplemental Figure 4), and a dose response curve for the most effective siRNA demonstrated an IC50 of less than 25 nM for hnRNP L siRNA with, again, no effect observed for hnRNP A2/B1 siRNA (Supplemental Figure 5, A and B). Lastly, several stable cell lines were produced from hnRNP L and hnRNP A2/B1 shRNA. All of the hnRNP L shRNA cell lines demonstrated a significant effect on the ratio of caspase-9a/9b mRNA (Supplemental Figure 5C). In contrast, none of the hnRNP A2/B1 shRNA cell lines presented with an increase in the ratio of caspase-9a/9b mRNA (Supplemental Figure 5D). As caspase-9a and 9b are transcribed from the same gene and possess the same 5′UTR and the same 3′UTR sequences, the effect of hnRNP L downregulation is likely attributable to the regulation of the alternative splicing of caspase-9 and not to effects on transcription, translation, or mRNA stability. Specifically, transcriptional activation/repression did not account for the changes in the C9a/9b mRNA ratio, as both were derived from the same promoter and actinomycin D treatment did not affect the caspase-9a/9b mRNA ratio (data not shown). The observed effect of hnRNP L siRNA also cannot be attributed to translation, as the observed changes in the ratio of caspase-9a/9b mRNA were mirrored at the protein level. There is the possibility that changes in mRNA stability played a role in our observations, as hnRNP L has been shown to regulate the stability of specific mRNAs (24, 25), but the half-lives of caspase-9a and -9b mRNA were previously shown by our laboratory to be unaffected by agonists (17). Furthermore, treatment of A549 control shRNA and A549 hnRNP L shRNA cell lines with actinomycin D demonstrated no significant difference in the decay rates (e.g., _t_ν) of caspase-9a and caspase-9b mRNAs in the presence or absence of hnRNP L (data not shown). This finding suggests that hnRNP L does not regulate the mRNA stability of either splice variant, although known caveats in the use of actinomycin D make definitive conclusions difficult. Overall, the data support the conclusion that hnRNP L and not hnRNP A2/B1 acts as a repressor of the inclusion of the exon 3,4,5,6 cassette of caspase-9 via manipulation of the alternative splicing of caspase-9 pre-mRNA.
To determine whether this mechanism demonstrated translatability in NSCLC, hnRNP L siRNA was again used in H838 and H2030 cells. The role of hnRNP L was further corroborated in these NSCLC cell lines, as downregulation of hnRNP L by siRNA produced a concomitant increase in the ratio of caspase-9a/9b mRNA for H838 (2.0 ± 0.13 for siControl, 4.1 ± 0.09 for sihnRNP L) and H2030 (2.1 ± 0.13 for siControl, 3.7 ± 0.22 for sihnRNP L) (Supplemental Figure 6, A and B).
To demonstrate the specificity of hnRNP L in regulating the alternative splicing of caspase-9, other major factors of apoptosis were also examined (e.g., caspase-8, caspase-2, and Bcl-x) using quantitative/competitive RT-PCR. Downregulation of hnRNP L with siRNA demonstrated no effect on the alternative splicing of caspase-8, caspase-2, and Bcl-x (Supplemental Figure 7). Thus, the effect of downregulating hnRNP L is specific for regulating the alternative splicing of caspase-9, and not a generalized effect on pre-mRNA processing.
Since siRNA and shRNA can have numerous biological and sequence-specific effects, the reciprocal experiment was also undertaken in which low ectopic expression of hnRNP L in conjunction with the caspase-9 minigene was utilized. Transfection of A549 cells with hnRNP L (low ectopic expression) had the opposite effect of hnRNP L siRNA, which induced a moderate but significant reduction in the ratio of caspase-9a/9b minigene mRNA (Figure 6A). Note that the minigene caspase-9a/9b mRNA ratio was slightly higher in A549 cells (2.7–3.2) compared with the endogenous caspase-9a/9b ratio (1.9–2.2). To demonstrate translatability, H2030 and H838 cells were again utilized, and low ectopic expression of hnRNP L demonstrated the same effect on lowering the ratio of the caspase-9a/9b minigene mRNA (Figure 6, B and C).
Low ectopic expression of hnRNP L decreases the caspase-9a/9b splicing ratio. (A) A549, (B) H838, and (C) H2030 cell lines were transfected with wild-type hnRNP L (WT-hnRNP L) (0.25 μg) in conjunction with caspase-9 minigene (C9 WT MG) (0.25 μg) or with caspase-9 minigene (C9 WT MG) (0.25 μg) in conjunction with empty vector (EV) (0.25 μg) for 24 hours. Total RNA was extracted and analyzed by competitive/quantitative RT-PCR for caspase-9 minigene splice variants. Data are n = 4 from 2 separate occasions. Note that the ratio of caspase-9a/9b minigene mRNA tended to present with a slightly higher ratio than endogenous caspase-9a/9b mRNA.
hnRNP L regulates the AIG and tumorigenic capacity of NSCLC cells via the pre-mRNA processing of caspase-9. Modulation of the levels of splice variants of caspase-9 had dramatic effects on AIG and the tumorigenic capacity of NSCLC cells. Thus, we hypothesized that hnRNP L plays a major role in maintaining the transformed phenotype of NSCLC cells through modulation of the pre-mRNA processing of caspase-9. To test this hypothesis, multiple clones of A549 cells stably expressing hnRNP L shRNA were produced (Figure 7A and Supplemental Table 3). As with the studies utilizing caspase-9b shRNA cell lines, the first physiologic parameter examined was AIG. Stable expression of hnRNP L shRNA abolished the ability of A549 cells to grow in soft agar (Figure 7, B and C). More importantly, low ectopic expression of caspase-9b cDNA (e.g., the caspase-9a/9b mRNA ratio dropped from 3.8 to 1.6) in the hnRNP L shRNA cell lines rescued the loss of AIG in A549 cells (Figure 7, B and C). These effects did not require stable expression and were not due to integration artifacts, as short-term expression of hnRNP L shRNA or use of stable batch cell lines produced comparable results (data not shown). Furthermore, the effect on AIG induced by downregulation of hnRNP L was specific for the loss of caspase-9b, as ectopic of expression of apoptosis inhibitory factors CrmA and Bcl-x(L) did not rescue the phenotype (Figure 7D). Lastly, the stable re-expression of shRNA-resistant hnRNP L also rescued the loss of AIG induced by the loss of hnRNP L shRNA, demonstrating specificity for the loss of hnRNP L in both the RNA splicing and biological mechanisms (Figure 7E).
hnRNP L regulates the tumorigenic capacity in A549 cells via the alternative splicing of caspase-9. (A) Total RNA from the designated cell lines was analyzed for caspase-9 splice variants. (B) Colony formation assays in soft agar for the designated cell lines. (C) Quantization of the number of colonies for the indicated clonal cell lines. n = 6; error bars represent SEM. *P < 0.005, **P < 0.001 versus A549 vector and shRNA control; Student’s t test. (D) Top: Total protein from the listed cell lines was subjected to SDS-PAGE analysis and immunoblotted for hnRNP L, Bcl-xL, CrmA, and β-actin. Bottom: Colony formation assays in soft agar for the cell lines shown at top. n = 9, error bars represent SD. A significant effect was observed compared with vector control cells (e.g., P < 0.005, Student’s t test). (E) Top: Total RNA from the listed clonal cell lines and analyzed for caspase-9 splice variants. Bottom: Quantization of the number of colonies for the indicated stable batch culture cell lines. n = 6; error bars represent SEM. #P < 0.05 compared with A549 + hnRNP L shRNA; Student’s t test. RM, resistant mutant. (F) The designated cell lines were injected subcutaneously into SCID mice. Tumor volume was measured at the end of 28 days. Error bars indicate SEM. (G) H&E stain of the tumors presented in D.
These observations translated to the NSCLC cell lines, H838 and H2030 cells, as no stable clones of either cell line were obtained for hnRNP L shRNA, in contrast to control shRNA and null lentivirus. Thus, both H838 and H2030 completely lost clonogenic capacity in the absence of hnRNP L, which correlated in the induction of apoptosis as assayed by PARP cleavage (e.g., caspase activation) (data not shown). Importantly, stable expression of caspase-9b cDNA rescued this phenotype, allowing for stable clones to be obtained for hnRNP L shRNA (data not shown). Thus, the loss of clonogenic capacity by stable expression of hnRNP L shRNA occurred at least in part via loss of caspase-9b expression. Therefore, the biological effects of hnRNP L downregulation and subsequent modulation of the pre-mRNA processing of caspase-9 translates to other NSCLC cell lines.
The tumorigenic capacity of these cell lines was also characterized using A549 xenograph tumor formation in severe combined immunodeficiency mice. Figure 7F shows that stable downregulation of hnRNP L in A549 cells led to a complete loss of tumor formation (0/8 cell injections). Pathological analysis confirmed the complete loss of tumor formation for hnRNP L shRNA cell lines. Comparable to the AIG effects, ectopic expression of caspase-9b completely rescued the loss of tumor formation induced by downregulation of hnRNP L (7/7 xenographs). Furthermore, pathological analysis of the hnRNP L shRNA/caspase-9b cell line demonstrated that ectopic expression of C9b also rescued the classification/grade of the hnRNP L shRNA cell line (Figure 7G). Overall, these data demonstrate that the loss of hnRNP L dramatically alters the tumorigenic capacity of NSCLC cells. Furthermore, these data demonstrate that hnRNP L shRNA predominantly affects the tumorigenic capacity via manipulation of the pre-mRNA processing of caspase-9.
The phosphorylation of hnRNP L on Ser52 regulates the alternative splicing of caspase-9 in NSCLC. The expression of hnRNP L was not increased in NSCLC cells, and so hnRNP L expression did not correlate with a low ratio of caspase-9a/9b mRNA (Figure 8A). This disparity was further enhanced by the observation that neither downregulation of hnRNP L nor ectopic expression of hnRNP L in immortalized HBEC-3KT cells affected the inclusion/exclusion of the exon 3,4,5,6 cassette of caspase-9 in the same manner as NSCLC cells (Figure 8, B and C).
The role of hnRNP L in regulating the alternative splicing of caspase-9 in non-transformed human bronchial epithelial cells. (A) Total protein lysates from A549, H838, H2030, and HBEC-3KT cell lines were subjected to SDS-PAGE analysis and immunoblotted for hnRNP L and β-actin. (B) HBEC-3KT cells were transfected with 100 nM control siRNA or 100 nM hnRNP L SMARTpool siRNA for 48 hours. Total RNA was isolated and analyzed by competitive/quantitative RT-PCR for caspase-9 splice variants. Simultaneously, protein lysates were also produced, subjected to SDS-PAGE, and immunoblotted for hnRNP L and β-actin. Data are n = 4 from 2 separate occasions. (C) HBEC-3KT cell lines were transfected with either wild-type hnRNP L (0.25 μg) in conjunction with caspase-9 minigene (0.25 μg) or caspase-9 minigene (0.25 μg) in conjunction with empty vector (0.25 μg) for 24 hours. Total RNA was extracted and analyzed by competitive/quantitative RT-PCR for caspase-9 minigene splice variants. Data are n = 3 on 2 separate occasions. Of note: the ratio of caspase-9a/9b minigene mRNA tended to present with a slightly higher ratio than endogenous caspase-9a/9b mRNA.
The above results suggested several possibilities for the contrasting findings in NSCLC cells versus non-transformed cells such as mislocalization (26), post-translational modifications of hnRNP L (27, 28), or a combination of mislocalization and post-translational modifications, which would modify the repressing capabilities of hnRNP L. To date, no confirmed phosphorylated site(s) have been established for hnRNP L functionality. Supplemental Figure 8 presents data that ruled out the mislocalization of hnRNP L; no difference was observed in cellular distribution between HBEC-3KT cells and several NSCLC cell lines. The phosphorylation state of hnRNP L between these cell lines was then examined using specific phosphorylated residue antibodies, and A549 cells demonstrated a dramatic enhancement of phosphorylated hnRNP L compared with HBEC-3KT cells (Figure 9A). Specifically, hnRNP L presented with a dramatic 10-fold increase in phospho-threonine, along with a marked increase in both serine (4-fold) and tyrosine (2-fold) phosphorylation in A549 cells compared with the non-transformed HBEC-3KT cells. To determine whether this observed phosphorylation demonstrated translatability in NSCLC, hnRNP L IP was used in H838 cells, and this NSCLC cell line also presented with a significant increase in serine and threonine phosphorylation (data not shown). Thus, an enhanced phosphorylation state of hnRNP L was observed in NSCLC cells versus non-transformed cells.
Ser52 of hnRNP L is hyperphosphorylated in NSCLC cells and regulates the alternative splicing of caspase-9. (A) A549 and HBEC-3KT cell lines were seeded at the same confluency and in the same culture media 24 hours before IP. IP hnRNP L was resolved by SDS-PAGE and immunoblotted with phospho-specific and hnRNP L antibodies. (B) A549 cells were transfected with WT-hnRNP L (L-WT) (1 μg), Ser52Ala hnRNP L (S52-A) (1 μg), or Ser52Asp hnRNP L (S52-D) (1 μg). Total RNA was analyzed for caspase-9 splice variants. n = 4 from 3 occasions. (C) A549 cells were transfected with WT-hnRNP L, S52-A, or S52-D. Protein lysates were subjected to SDS-PAGE and immunoblotted for myc-tag and β-actin. Empty vector showed no expression of a myc-tagged protein. (D) A549 and HBEC-3KT cell lines seeded at the same confluency and in the same culture media for 24 hours before IP. Cell lines were transfected with WT-hnRNP L (1 μg) or S52-A (1 μg). Ectopically expressed hnRNP L was IP with c-myc tag Ab, resolved by SDS-PAGE, and immunoblotted with anti–phospho-serine and anti–c-myc tag antibodies. (E) Protein lysates were subjected to SDS-PAGE and immunoblot for phospho-Ser52 hnRNP L and hnRNP L. Phospho-Ser52 antibody for hnRNP L was validated by ELISA, hnRNP shRNA samples, and lack of identifying the Ser52Ala mutant of hnRNP L. (F) Colony formation assays in soft agar. n = 6; error bars represent SEM. *P < 0.005, A549 + S52-A hnRNP L + C9b ectopic versus A549 + S52-A hnRNP L.
Once hnRNP L was determined to be hyper-phosphorylated in a cancer-specific manner, we analyzed the amino acid sequence for hnRNP L for phosphorylation sites by mitogenic, cell survival, and oncogenic kinases. Several mass spectrometry databases were also consulted for references to identified/verified phosphorylated amino acids for hnRNP L. Supplemental Table 4 designates the possible phosphorylated sites for hnRNP L as predicted by PhosphositePlus, a phosphorylation site determination database (Cell Signaling Technology) (29), Human Protein Reference Database resource (30), Scansite (Massachusetts Institute of Technology) (31), and UniProtKB/Swiss-Prot (32). To determine which of these residues of hnRNP L regulated the exclusion of the exon 3,4,5,6 cassette of caspase-9, site-directed mutagenesis was used to convert relevant serines and threonines to alanine, and tyrosines to phenylalanine. A549 cells were transiently transfected (>50% transfection efficiency) with each mutant or wild-type hnRNP L, and the effect on the alternative splicing of endogenous caspase-9 was assayed by competitive/quantitative RT-PCR. Only ectopic expression of hnRNP L (Ser52Ala) induced a significant increase in the splicing ratio of caspase-9 in comparison with ectopic expression wild-type hnRNP L and empty vector controls (Figure 9B; Supplemental Table 4). To verify this result and rule out structural problems caused by mutation of this residue, a phosphomimic mutation, hnRNP L (Ser52Asp), was created. In contrast to the Ser52Ala mutation, transfection of hnRNP L (Ser52Asp) in A549 cells did not significantly affect the endogenous splicing ratio of caspase-9 compared with ectopic expression of wild-type hnRNP L, suggesting that either the levels of phosphorylated hnRNP L in NSCLC cells are sufficient to affect limiting levels of endogenous caspase-9 pre-mRNA, or that this mutation cannot appropriately mimic the phosphorylation of Ser52 (Figure 9B). Equivalent expression was verified by Western immunoblotting for the myc-tag associated with ectopically expressed hnRNP L (Figure 9C). The latter hypothesis is likely correct as ectopic expression of hnRNP L (Ser52Asp) in HBEC-3KT cells was not sufficient to reduce the endogenous caspase-9a/9b mRNA ratio (Supplemental Figure 10). Both phosphorylation mutants of hnRNP L also localized to only the nucleus (data not shown).
To demonstrate that phosphorylation on Ser52 was responsible for the serine hyper-phosphorylation of hnRNP L in NSCLC cells, wild-type hnRNP L and the Ser52Ala mutant of hnRNP L were transfected into A549 and HBEC-3KT cells, followed by specific IP of ectopically expressed/myc-tagged hnRNP L. Whereas ectopically expressed wild-type hnRNP L in A549 cells demonstrated a more than 3-fold increase in serine phosphorylation compared with ectopic expression in HBEC-3KT cells (in complete agreement with the endogenous findings for hnRNP L presented in Figure 9A), the Ser52Ala mutant of hnRNP L demonstrated a dramatic reduction in serine phosphorylation comparable to non-transformed HBEC-3KT cells (Figure 9D). To further demonstrate that hnRNP L is phosphorylated on Ser52 in a cancer-specific manner, we created a custom phospho-specific Ser52 hnRNP L antibody. Whole-cell lysates from A549, H838, H2030, and non-transformed HBEC-3KT cell lines were used to examine phospho-Ser52 state of hnRNP L by Western immunoblot analysis. All 3 cell lines demonstrated the higher expression of phospho-Ser52 hnRNP L compared with non-transformed HBEC-3KT cells, which demonstrated no discernible phospho-Ser52 hnRNP L (Figure 9E). Thus, hnRNP L is specifically phosphorylated on Ser52 in NSCLC cells, and the phosphorylation state of this residue of hnRNP L regulates the pre-mRNA processing of caspase-9.
To examine whether Ser52 of hnRNP L played a role in AIG of NSCLC cells, the hnRNP L (Ser52Ala) mutant and wild-type hnRNP L were stably expressed as batch cultures of A549 cells. Analogous to transient expression of the hnRNP L Ser52Ala mutant, stable expression of the hnRNP L Ser52Ala mutant induced an increase in the caspase-9a/9b mRNA ratio from 2.0 ± 0.13 to 3.2 ± 0.10. Stable transfection of the hnRNP L (Ser52Ala) mutant dramatically decreased the AIG ability of A549 cells in comparison with stable expression of wild-type hnRNP L (Figure 9F). Furthermore, re-expression of caspase-9b in the same cell lines completely rescued the effect of the hnRNP L (Ser52Ala) mutant on AIG (Figure 9F). Therefore, and in an analogous fashion to hnRNP shRNA, the phosphorylation of hnRNP L on Ser52 regulates the ability of NSCLC cells to grow in an anchorage-independent manner via the pre-mRNA processing of caspase-9.