MSLN overexpression is a hallmark of the majority of PDA cells as shown by us and numerous investigators ( and ). In this current study, we report the in vitro exploitation of the naturally occurring transcriptional pathway that leads human pancreatic cancer cells to overexpress MSLN. The marked reduction of luceriferase activity (protein synthesis) in two MSLN+
pancreatic cancer cell lines (over 95% in both instances) following transfection with a MSLN/DT-A DNA construct establishes a novel paradigm for specifically targeting toxic molecules to pancreatic tumor cells. Since MSLN overexpression has been linked to various solid tumor types,6,16–18,32
we predict that these findings can be extended to other forms of cancer, as has already been accomplished in ovarian cancer cells by Sawicki and colleagues.
We demonstrate DT-A inhibition of protein synthesis and cell survival of MSLN+
pancreatic cancer cells. Theoretically, a single molecule of DT-A toxin is able to kill a host cell by inhibiting protein synthesis.20,33
Because the DT-B subunit is not included, the DT-A released from the dead cells is not able to enter surrounding cells.21
This strategy of targeting protein synthesis in pancreatic cancer cells is especially promising because the downstream functional effects of the common mutational events in pancreatic cancer, K-ras activation and inactivation of two tumor suppressors (p53 and Rb), have been previously shown to increase protein synthesis in other experimental models. These studies have shown that K-ras and the p53 and Rb pathways co-regulate the protein synthesis network. In pancreatic cancer, the oncogene K-ras is constitutively activated (i.e., turning on protein synthesis), while the tumor suppressor pathways, p53 and Rb (through p16 silencing), are inactivated and thus can not regulate protein synthesis.4,34–38
Loss and gain of function of these proteins would in theory affect the regulatory circuits of the pol III network that controls protein synthesis. Further, it has been shown that mTOR is a key regulator of protein synthesis and is overactive in pancreatic cancer cells.39
mTOR interacts with elongation factor 2 which is directly targeted by DT-A.33,40
Thus, targeting protein synthesis via pancreatic cancer cell-specific expression of DT-A DNA is a logical approach of targeting a possible collaborative functional outcome of the most frequent mutations (i.e., K-ras
) found in pancreatic tumors.4
In this study, we utilized a nanoparticulate delivery system that has already been translated in vivo for prostate cancer,22
and more recently, ovarian cancer. This modern mode of gene delivery is exciting because thorough investigation and refinement of these polymers have shown them to be safe, effective and biodegradable vehicles for suicide DNA delivery in vitro and in vivo.23
Using polymers to deliver suicide DNA has numerous advantages over more conventional viral-based vectors of gene therapy. Most importantly, this DNA delivery system is easy to produce, stable and highly efficient at delivering large amounts of DNA.21
One limitation to the therapeutic approach of using DT-A to kill cancer cells is the possibility of a ‘leaky’ promoter, and subsequent unwanted cell death in normal tissues. Further, in characterizing MSLN-reactive antibodies, Onda M. et al. detected no expression of MSLN in normal tissues including liver, lung, ovarian stroma, brain, breast, uterus (endometrium, myometrium), placenta and kidney tissues. Yet, MSLN expression was detected in the cancerous tissue, except for the peritoneal mesothelium (on uterus), pleural mesothelium.41
Thus, even with a cancer-specific promoter, normal cell death may occur when cellular mechanisms that control expression of toxic genes are not tightly controlled. Previous work has shown that the use of a dual regulatory system that combines transcription control with site-directed DNA recombination, successfully and specifically controls the expression of DT-A in cancer cells.27,42
We plan to use this strategy to assure pancreatic cancer specific targeting by utilizing cancer specific enhancer elements from the MSLN promoter14
and combining this with the Prostate Stem Cell Antigen (PSCA) promoter that would be specifically active in pancreatic cancer cells.43
If additional pre-clinical studies show errant DT-A expression, inclusion of this dual regulatory strategy into DNA constructs could be easily adopted.
Future studies will explore the efficacy of this therapeutic strategy in the K-ras/p53 pancreatic mouse model.44
We have recently determined that tumors in these mice express MSLN (our unpublished findings). Currently, we are also attempting to optimize the MSLN promoter by incorporating recent advances in our understanding of MSLN transcriptional regulation.14
Ultimately, testing pancreatic tumors post-operatively for MSLN expression () may stratify patients for potent targeted nanotherapy. Thus, nanotherapy may be a safe and effective way to manage pancreatic cancer patients in the neoadjuvant and adjuvant settings alone, or in combination with other targeted therapies.