This report demonstrates that inhibition of AKT phosphorylation with the cell-permeable peptide TAT-CTMP4 results in a dose-dependent induction of apoptosis in model pancreatic adenocarcinoma. This appears to be an effect that is limited to specific cell types, as PBMC, fibroblasts, and some adenocarcinomas are unaffected by treatment with this peptide. Additionally, these data support the concept of utilizing biologic therapy (AKT inhibition) combined with chemo- or radiotherapy to further augment an anti-tumor response in resistant malignancies.
In this report, we reasoned that the highly structured, yet functionally uncharacterized region of CTMP was responsible for the observed AKT-inhibitory activity of the full length protein. Based on criteria presented by Hemmings, and the predicted secondary structure elements of the N-terminal domain of CTMP, we designed four TAT-conjugated peptides and screened them for their ability to induce pancreas adenocarcinoma cell apoptosis. The C-terminal most peptide (TAT-CTMP4, encompassing amino acids 94–117 of the full length protein) was found to cause significant pancreatic adenocarcinoma cell apoptosis. This peptide was predicted to have a helical structure and substitution of two bulky, aromatic residues on one face of the helix with alanines resulted in an inactive variant peptide. To our knowledge, this is the first report that a short peptide from CTMP can recapitulate the AKT-inhibitory activity of the full length protein.
AKT itself is rarely mutated in pancreatic adenocarcinoma. Ras
mutations, however, are among the earliest and most common genetic mutations in pancreatic cancer (16
). Specifically, activating K-ras
mutations are present in nearly all pancreatic adenocarcinomas. These activating K-ras
mutations result in the phosphorylation of membrane phosphatidylinositol which subsequently recruits PH domain-containing proteins, such as AKT, to the plasma membrane. This leads to the phosphorylation of AKT and the subsequent downstream activation of pro-survival signals (30
). Taken together, these observations suggest that targeting AKT may provide an important checkpoint in a multimodal approach to therapy. For these reasons, we reasoned it is rational to further develop this therapeutic strategy (inhibiting AKT phosphorylation with CTMP) in pancreatic cancer.
AKT is involved in a myriad of cell signaling pathways that grossly relate to cell survival, metabolism, and growth. Inhibition of this kinase has several, potentially beneficial effects. For instance, inhibiting the AKT-mediated phosphorylation of Bad would limit its sequestration and promote Bcl-2 activation leading to apoptosis (31
). Similarly, the AKT-mediated inhibition of GSK-3β may have therapeutic implications; GSK-3β is a core component of two pathways involved in cell fate determination and morphology: Wnt and Hedgehog. Both or these pathways are well characterized mediators of several cancers (33
It is important to distinguish this approach from a PI3K-targeted therapy. Although much enthusiasm exists for PI3K-targeted therapeutics, there also exists a potential for broad-based effects due to the wide-ranging intracellular actions of PI3K. In contrast, AKT, a downstream effector, has a smaller scope of action and may be better suited as a therapeutic target .
Though more thorough testing is needed to fully evaluate the potential toxicity of a CTMP-based therapeutic, the intracellular signaling profile, as measured by phospho-protein measurements, is encouraging. Namely, the absence of any change in the phosphorylation state of IKB suggests that the pro-inflammatory effectors of the NF-kB pathway are not activated as a result of this Akt-targeted therapy. Indeed, the high degree of specificity of CTMP for AKT (shown by us, Hwang et al and Maira et al) suggests that broad-based toxicity is unlikely. Recently, Ono et al have shown that CTMP may have cell context dependent activity as an Akt agonist (34
). Our data suggest that this is not the case in pancreas or pancreas adenocarcinoma, however, it will be important to examine this in detail. Because our approach uses a single linear epitope from the endogenous protein, it is not likely subject to post-translational modifications that may alter its activity.
TAT-CTMP4 was effective in the treatment of both human and murine pancreatic adenocarcinoma. This phenomenon was observed in the setting of only a 68% sequence homology between the murine and human sequences. This suggests that TAT-CTMP4 treatment may be broadly applied to pancreatic cancers and may imply applicability to other forms of malignancies in which the AKT survival pathway is highly active, including cancers of the ovary, breast, lung, thyroid, and gastrointestinal tract (35
) as well as our demonstration of activity in cancers of the kidney, colon and esophagus. Importantly, the residues mutated to make the inactive peptide are also conserved between murine and human systems.
The data presented here further suggest that radiation therapy may be useful to sensitize pancreatic malignancies to biologic therapy with TAT-CTMP4, which could be directly injected following irradiation of metastatic lesions, for example. This may facilitate a new role for radiation therapy in the treatment of pancreatic neoplasms.
It should be stressed that while our overall objective is to eradicate pancreas cancer with this biologic agent or a combination of conventional and biologic agents, such a therapy may also serve to downsize patients’ tumors such that they become candidates for curative resection. Alternatively, a targeted therapy based on our CTMP compound may be highly useful in the treatment of focal recurrences, especially when combined with gemcitabine or radiotherapy.
We recognize that this treatment modality, directly translated, may be of most interest in other directly-injectable malignancies (such as extremity sarcoma, malignant skin lesions, etc.) that can be easily accessed and do not require systemic targeting. With that said, it may be possible to inject TAT-CTMP4 into pancreatic malignancies via CT-guidance (metastatic disease) or with Endoscopic Ultrasound (EUS). Furthermore, since activity appears high in pancreatic tissue, there may exist a role in infiltrating the surgical bed (at the time of resection) with such a therapeutic given the high rates of local recurrence in surgical patients (38
Alternatively, one may envision a therapeutic strategy that combines the pro-apoptotic therapy of CTMP with other inducers of apoptosis. Combination strategies could include TAT-BIM which targets intrinsic apoptotic pathway via antagonizing anti-apoptotic Bcl-2 family members and similarly induces apoptosis in these model systems (22
). An additional pathway may also include PD98059, an inhibitor of the MAP / ERK kinase pathway. Targeting multiple pathways may result in both greater levels of apoptosis as well as the favorable prospect of utilizing lower doses of chemotherapeutic drugs. This is especially true with the highly toxic chemotherapeutic gemcitabine.
Our current goal is to engineer a compound that specifically targets pancreas cancer with the pro-apoptotic peptide CTMP4. Possible targeting strategies that may facilitate delivery of CTMP4 to pancreas cancer may include coupled ligands to any number of tumor-associated cell surface targets (39
). We are currently synthesizing such agents and screening their ability to recapitulate the TAT-CTMP4-mediated induction of apoptosis in these model systems.
In summary, we have generated a novel therapy for the treatment of pancreatic adenocarcinoma that directly targets AKT (TAT-CTMP4). This biologic agent induced apoptosis in all pancreas adenocarcinoma cell lines and several other adenocarcinoma cells in vitro. This effect is limited in scope as important non-transformed cell types including PBMCs and fibroblasts were refractory to treatment. Certain adenocarcinomas were also resistant to CTMP-induced apoptosis. This treatment significantly augmented the effect of conventional therapies (chemo- and radiotherapy) in vitro and in vivo. These data suggest that directly inhibiting AKT with CTMP is a promising strategy for the treatment of pancreatic adenocarcinoma.