The importance of the presented study lies in several key findings. First, our mechanistic knowledge of how the alternative splicing of caspase 9 is regulated has been expanded by our identification of a novel RNA cis-element via which SRSF1 (ASF/SF2) enhances the inclusion of the exon 3,4,5,6 cassette. Furthermore, SRSF1 was shown to specifically interact with this RNA cis-element, and regulate the alternative splicing of caspase 9 via this novel RNA cis-element. Second, we demonstrate that the alternative splicing of caspase 9 is a relevant therapeutic target as demonstrated by direct manipulation of this splicing cascade having significant effect on the sensitivity NSCLC cells to clinically relevant chemotherapeutics. Lastly, one of the major findings of the report is our data showing that the synergism of erlotinib combination therapy is in part via modulation of the alternative splicing of caspase 9.
In regards to the RNA cis
-element that specifically interacts with SRSF1, a purine-rich RNA cis
-element was identified in intron 6, 24 bp downstream of the 5′ splice site of exon 6. The position of this RNA cis
-element makes logical sense as it is localized near a juxta-exon for the large exon 3,4,5,6 cassette. We had initially anticipated that regulatory elements for the exon 3,4,5,6 cassette would be positioned in or near exon 3 and 6, and indeed, this study showed that an intronic splicing enhancer (now termed C9-I6/ISE) was immediately downstream of exon 6. There is still a possibility that C9-I6/ISE is only one of several required splicing enhancers for SRSF1 within the exon 3,4,5,6 cassette, and indeed, mutation of one other possible RNA cis
-element for SRSF1 interaction in exon 6 had a significant, albeit smaller effect on the caspase 9a/9b mRNA ratio. Still, C9-I6/ISE is the strongest enhancer element identified to date, and mutation of this RNA cis
-element produced the same results as downregulation of SRSF1 by siRNA (15
). This is another key indicator that C9-I6/ISE is the major enhancer element for inclusion of the 3,4,5,6 cassette. Furthermore, our studies demonstrate that SRSF1 specifically binds C9-I6/ISE, and wild-type SRSF1 as well as the phospho-mutants of SRSF1 could not affect the ratio of minigene caspase 9a/9b mRNA when C9-I6/ISE was mutated. Therefore, the culmination of these date demonstrate that C9-I6/ISE is the major splicing enhancer for the inclusion of the exon 3,4,5,6 cassette via specific interaction with SRSF1.
As more data is accumulated on the regulation of the exon 3,4,5,6 cassette of caspase 9, a complex and novel mechanism is beginning to emerge. For example, we recently demonstrated that an exonic splicing silencer (ESS) was located in exon 3 termed C9/E3-ESS. Indeed, we further showed that the RNA trans-factor, hnRNP L, bound this sequence in non-small cell lung cancer (NSCLC) cells to repress the inclusion of the exon 3,4,5,6 cassette into the mature transcript. In this same report, we addressed the possibility that this was an oversimplification, and indeed, we identified two additional ESS sequences in the exonic cassette, both in exon 4. These sequence share homology to the C9/E3-ESS sequence suggesting that hnRNP L may also associate with these sequences to repress the entirety of the cassette. This repression by hnRNP L activated by phosphorylation along with the inactivation of SRp30a by phosphorylation on ser199, 201, 227, 234 bound to exon 6 is a logical mechanism to rapidly regulate the alternative splicing of caspase 9. Overall, the additional regulatory RNA cis-elements identified in these studies suggest a complex mechanism involving a number of RNA cis-elements along the exon 3,4,5,6 cassette required for both inclusion and exclusion (repression). For example, repression of the exonic cassette requires not one, but a number of exonic splicing silencers bound to inhibitory RNA trans-factors (e.g. phosphorylated hnRNP L). Hence, the default splicing paradigm is inclusion of the exonic cassette as long as SRSF1 is expressed and not phosphorylated on ser199, 201, 227, 234, peripheral phosphorylation sites to the standard RS domain of the RNA trans-factor. Taken together with our recent reports on SRSF1 and hnRNP L in this alternative splicing mechanism, these new results suggest that RNA trans-factors not only play constitutive roles in RNA splicing, but also activated roles in modulating alternative splicing induced by post-translational modifications in contrast to simple expression gradients.
In regards to biology, the role of SRSF1 in this mechanism is translatable to more than one NSCLC cell line as well as other cell types. Our laboratory previously published that downregulation of SRSF1 by siRNA induced a dramatic decrease in the caspase 9a/9b mRNA ratio of A549 cells (15
). Essentially, the inclusion of the exon 3,4,5,6 cassette was abolished into the mature transcript. Here, we expanded this early study and demonstrated that downregulation of SRSF1 in the NSCLC cell lines, H2030 and H838 cells, the cervical cancer cell line, HeLa cells, and in the non-transformed immortalized human bronchial epithelial cell line, HBEC-3 cells, also lowered the caspase 9a/9b mRNA ratio. Whereas the effect of SRSF1 in the NSCLC cell lines and HBEC-3 cells was not unexpected, the result in HeLa cells was surprising due to a previous report by Krainer and co-workers (30
). Specifically, downregulation of SRSF1 was reported to have no effect on the alternative splicing of caspase 9 (30
). Unfortunately, our findings are not in agreement. This may be due to the extent of downregulating SRSF1 in HeLa cells or other technical differences. During the preparation of this report, an additional study published by Silver and co-workers, which also reported that the downregulation of SRSF1 affected the alternative splicing of caspase 9 in HeLa cells, but in this case, the conclusion was an increase in the caspase 9a/9b mRNA ratio (31
). This difference is likely due to this report examining only the levels of caspase 9a for their conclusions (31
). Regardless of these contrasting findings between research groups, all of the data reported in this manuscript demonstrate that SRSF1 is a required RNA trans
-factor for the inclusion of the 3,4,5,6 exon cassette of caspase 9 pre-mRNA in all cell lines examined to date, which is further supported by the corroborating effect observed by mutation of the SRSF1 binding site in the caspase 9 minigene (e.g. also lowered the caspase 9a/9b ratio).
Also in this study, we continued to address the biological consequences of a dysregulated caspase 9a/caspase9b ratio, and demonstrate that this distal splicing mechanism was related to the sensitivity of NSCLC cells to additional types of chemotherapies beyond EGFR inhibitors like erlotinib. For example, C9b siRNA induced a 2.7-fold increase in the caspase 9a/9b mRNA ratio, which translated to a significant decrease in the IC50 of DNA-damaging agents. Thus, the ratio of caspase 9a/9b mRNA may be a prognostic/predictive indicator of chemotherapy response to DNA-damaging agents. On the other hand, the ratio of caspase 9a/9b mRNA may not function as an indicator of response to classical cytotoxic agents such as paclitaxel. As this study shows, the shift in IC50 after either E4 treatment or 9b-si treatment in the case of Paclitaxel was significant, but not as nearly as dramatic when compared to the effects on the IC50 of DNR and Cp. This could be attributed to the mode of action for Pac versus the DNA damaging agents. For example, DNR sensitivity is highly linked to apoptosis capacity, thus manipulation of caspase 9 activity dramatically affecting the IC50 of this agent is logical. Regardless of the limited effect of the manipulating the caspase 9a/9b ratio on Pac, the effect on the IC50 of these chemotherapeutic agents translated to more than one NSCLC cell line suggesting a broad-based mechanism. Therefore, this study demonstrates that a single mechanism in these cells (e.g. the alternative splicing of caspase 9) has a large impact on the sensitivity of non-small cell lung cancer cells to several chemotherapeutic agents.
These findings have direct implications to the resistance observed in NSCLC patients to these compounds in the clinic. Specifically, our results suggest that direct manipulation of the caspase 9a/9b ratio would sensitize NSCLC tumors to already available chemotherapies. Furthermore, direct manipulation of this splicing event may allow for the use of one anti-cancer agent limiting side-effects and possibly toxic deaths as synergism between erlotinib and the DNA-damaging agent, daunorubicin, was lost by direct manipulation of the caspase 9a/b ratio. This is further highlighted as caspase 9b is only appreciably expressed in transformed cells, and hence, should have essentially no unwanted side-effects in patients. Currently, pre-clinical animal studies are being undertaken to assess the efficacy of this treatment regime.
In conclusion, this study links our previous findings on the phospho-status of SR proteins and the alternative splicing of caspase 9 during apoptotic signaling (15
) by establishing a mechanistic link between the SR proteins, SRSF1, and the alternative splicing of caspase 9 via a novel ISE in intron 6 that regulates the inclusion of the exon 3,4,5,6 cassette. These current studies also strongly suggest that the alternative splicing of caspase 9 is an important regulatory mechanism that influences the chemotherapy sensitivity of NSCLC cells. The direct manipulation of this mechanism with the help of ASROs, siRNA, and RNA scavengers gives us the promise of a new generation of molecular-therapeutic agents for NSCLC that sensitizes NSCLCs to a variety of chemotherapeutic agents with little or no unwanted side-effects.