K-Ras is oncogenically mutated in 90% of PDAC and has a critical role in the development and progression of this disease (3
). Since no anti-Ras therapies have reached success in the clinic, current efforts are focused on identifying key signaling pathways of Ras as possible targets for drug intervention (8
). Kinases are considered to be particularly tractable drug targets (4
), so identifying downstream kinases required for K-Ras-mediated transforming properties in PDAC might be a particularly appealing approach to novel PDAC therapeutics. Members of the oncogene family of Pim serine/threonine kinases are upregulated in many cancers (13
), have been shown to support cell growth and survival (13
), and are the subjects of a hunt for novel molecularly targeted therapeutics (13
). Our findings of Pim-1 protein expression being modulated by oncogenic K-Ras activity raise the possibility that Pim-1 levels may be a useful molecular marker of K-Ras activity in PDAC.
We show that Pim-1 protein is aberrantly expressed in a panel of PDAC cell lines as well as in tumors derived from PDAC patients compared with their matched normal tissues. Similarly, while our study was underway, a study conducted by Reiser-Erkan et al.
) showed upregulation of Pim-1 expression in malignant PDAC compared with benign and precancerous conditions of the pancreas. Therefore, we investigated whether Pim-1 might also contribute to PDAC growth transformation. Using shRNA-mediated knockdown of Pim-1 expression, we show here a requirement for Pim-1 in anchorage-dependent cell growth of PDAC cell lines known to be dependent on K-Ras. Similarly, Li et al.
) showed that suppression of endogenous Pim-3 resulted in decreased anchorage-dependent cell growth of PDAC cell lines. To further examine the mechanism of this growth inhibition in the PDAC cells infected with Pim-1 shRNAs, we analyzed the cell cycle and observed a 2-fold increase in the distribution of cells in G2
/M, suggesting cell cycle arrest as one mechanism for the differences in cell number. Additionally, we evaluated apoptosis as another potential explanation for the cell number differences in the Pim-1 knockdown cells. We observed modest increases in PARP and caspase-3 cleavage, suggesting that the differences could potentially reflect a greater susceptibility to apoptotic stimuli, which are supported by the decreased ratio of phosphorylated to total Bad protein (i.e. increased relative amounts of pro-apoptotic Bad protein in the presence of decreased Pim-1 expression). Mediators of this cell death other than Bad may be due to additional Pim-1 substrates such as apoptosis signaling kinase 1 (ASK1) or proline-rich Akt substrate 1 (PRAS1) (13
). Studies have shown that Pim-1 can inhibit apoptosis by direct phosphorylation of these substrates. Additionally, studies have shown that Pim-1 is involved in the localization and stability of various substrates (13
). Modulation of these processes may also lead to the susceptibility of apoptotic stimuli. Future studies should be conducted to evaluate how these various mediators of apoptosis can be regulated by Pim-1 expression and activity in PDAC.
To further consider the role of Pim-1 in PDAC growth transformation, we show here that suppression of endogenous Pim-1 expression decreased anchorage-independent growth of PDAC cells in the soft agar assay, which is routinely used to predict tumor growth in xenograft mouse models (25
). This decrease in growth may also be contributed to effects on the cell cycle and increased susceptibility to apoptotic stimuli due to Pim-1 suppression as discussed above. Our results are in agreement with recent studies in which expression of a Pim-1 kinase-dominant negative decreased tumorigenicity and chemoresistance of a PDAC cell line in a murine xenograft model (18
) and collectively suggest that high Pim-1 expression may be a marker of poor prognosis. Additionally, recent studies have demonstrated a role for Pim-1 in cell motility, migration and invasion in several cancer types including leukemia, prostate and tongue carcinoma (36
), but this has not been investigated in PDAC. We showed here that Pim-1 inhibition led to a significant decrease in PDAC cell invasion through Matrigel. Thus, Pim-1 contributes to invasive properties of K-Ras-dependent PDAC cell lines.
Finally, PDAC has been demonstrated to be insensitive to radiotherapy (31
), and K-Ras is an important contributor to radioresistance in this (28
) and other tumor types (30
). Here, we observed that suppression of Pim-1 expression decreased the intrinsic radioresistance of PDAC cells that we have shown previously are rendered resistant in part due to K-Ras activity (33
). These results are consistent with those of Peltola et al.
), who found that high Pim-1 kinase expression predicts poor radiation response in head and neck cancers. Together, these findings suggest that novel Pim-1 inhibitors may serve as radiosensitizers of PDAC and help to improve outcomes.
PDAC is a highly aggressive and lethal disease with dismal survival rates. In this report, we have demonstrated that Pim-1 kinase is downstream of K-Ras signaling and may serve as a molecular marker of oncogenic K-Ras activity. Overall, our studies indicate that Pim-1 plays a significant role in growth transformation, invasion and radioresistance of PDAC and is potentially a useful target for therapeutics in this disease. To date, there have been >50 Pim kinase inhibitors developed by research groups in both industry and academia (13
). One of the most potent of these, SGI-1776, impaired in vitro
and in vivo
growth of human leukemia cell lines and induced apoptosis in chronic lymphocytic leukemia and prostate cancer cell lines (42
). Results from these studies have led to a Phase I clinical trial to explore the safety of SGI-1776 for the treatment of refractory non-Hodgkin lymphoma and prostate cancer patients (http://www.clinicaltrials.gov
). Additionally, a recent study has identified 21 new substrates potentially targeted by Pim kinases (44
). Future studies will help to better understand the molecular mechanisms involving oncogenic K-Ras and Pim-1 signaling that are responsible for the aberrant growth properties of PDAC.