The role of the proto-oncogene c-Src in the progression of many tumors is well documented (19
). However, overexpression of wild-type Src is weakly oncogenic on its own (25
) and activating mutations are rare, limiting our understanding of Src in the development, maintenance, and progression of cancer (26
). Our results show that Src inhibition in pancreas cancer cells treated with dasatinib result in inhibition of cell proliferation, migration, invasion and anchorage independent growth, which directly correlate with a significant reduction in tumor growth in vivo
. We identified sensitive and more resistant pancreas cancer cell lines to dasatinib treatment to further elucidate the molecular mechanisms involved with Src inhibition. Src kinase inhibition with dasatinib blocks the activation of multiple downstream signal transduction pathways known to promote survival, angiogenesis, proliferation, motility, migration and invasion of tumor cells (31
) including AKT, STAT3, MAPK, JNK, FAK and paxillin, in sensitive cell lines (BxPC3).
Targeting signal transduction pathways is an effective strategy in many tumor types. However, activation of parallel signaling pathways can limit the efficacy of this approach, therefore, understanding the mechanisms of resistance to targeted agents remains critical to enhance their clinical usefulness. Resistance to dasatinib treatment appears to be related to lack of inhibition of STAT3 and MAPK signaling. STAT3 phosphorylation is inhibited with dasatinib treatment in sensitive pancreas cancer cell lines, but not in the more resistant PANC 1 cell line. Consistent with this finding, recently, reactivation of STAT3 signaling after Src inhibition has been shown in head and neck cancer (33
) and non-small cell lung cancer (34
). Somewhat selective to SFKs is their ability to activate STAT3, which leads to the activation of downstream targets including Bcl-XL, c-Myc (35
) and cyclin D1 (37
), resulting in increased cell survival, proliferation and tumor growth. Our results suggest that this compensatory pathway may account for the resistance to Src inhibition and implicates elevated STAT3 expression as a potential biomarker of this resistance. Furthermore, this data provides rationale to combine STAT3 inhibitors with dasatinib to improve clinical response in pancreas cancer.
Additionally, basal pAKT levels were also noted to be markedly elevated in the more resistant PANC1 cell line, as well as other less sensitive cell lines such as MiaPaca2 and AsPC1(data not shown), when compared with BxPC3 sensitive cells. Although, dasatinib treatment resulted in significant inhibition of pAKT activation in both PANC1 and BxPC3 cells, this increased basal pAKT levels may be another contributing factor towards the resistant nature of PANC1 cells and requires further investigation.
We also show for the first time that Src expression increases with progression of pancreas neoplasia from normal pancreas to chronic pancreatitis to PDA. In addition, the increase in Src expression with increasing tumor grade of PDAs is consistent with the role of Src activity playing an important role in cancer progression and metastasis (19
). Our finding that survival outcomes are significantly improved when Src expression is lower in the membranous compartment compared to the cytoplasmic compartment, suggests that cytoplasmic Src is weakly oncogenic and requires membranous localization to become activated and regulate important mechanisms of specific receptor pathways, stimulating oncogenesis. We have previously shown that activation of Src results in its translocation from the cytoplasm to the cell membrane where it activates TNF-α converting enzyme (TACE) to induce shedding EGFR ligands from the cell membrane and activate EGFR signaling (21
Interestingly, we also found that Src expression is increased in patients with chronic pancreatitis. Increased Src expression and activity are generally correlated with cancer progression and advanced malignancy. However these results implicate activation of Src at an earlier stage of pancreatic neoplasia and may suggest that patients with chronic pancreatitis may have a higher propensity toward malignant transformation depending on their levels of Src activation.
Dasatinib is a multi-targeted kinase inhibitor and it is likely that not all of its biological and molecular effects are due to Src inhibition alone. However, several lines of evidence suggest that the effects of dasatinib in pancreas cancer are related to Src kinase inhibition. Phosphorylation of Tyr416
in the kinase domain is a critical activation step in the regulation of Src tyrosine kinase activity (10
). We show Src phosphorylation at Tyr416
is inhibited with addition of dasatinib. In addition to our results with Src shRNA cells, these findings suggest that the predominant effects of dasatinib treatment is related to inhibition of Src kinase activity.
Inhibition of Src signaling has been shown to restore sensitivity to gemcitabine in cell lines derived from pancreas tumors. Similarly, silencing of FAK expression in PANC1 cells restores sensitivity to gemcitabine treatment (4
). Furthermore, inhibition of Src also has been shown to revert chemoresistance against 5-fluorouracil in human pancreas carcinoma cells (41
). Therefore, these results clearly implicate Src inhibition in restoring chemosensitivity and suggest a novel approach using dasatinib in combination with gemcitabine for the treatment of pancreas cancer.
There were no known genetic mutations that influenced cell sensitivity to dasatinib (). Inactivating p53
mutations, activating Ras
mutations (predominantly K-ras
) and Smad4/DPC4
mutations are commonly found in PDAs. We did not find any relationship between p53
status and sensitivity to dasatinib which is consistent with other studies in head and neck squamous cell carcinoma and non-small cell lung carcinoma (NSCLC) cells (42
In conclusion, we show that dasatinib inhibits pancreas tumor growth both in vitro and in vivo. Our results, for the first time, demonstrate that dasatinib is effective in inhibiting multiple signaling pathways involved in tumor growth, proliferation, angiogenesis, survival, motility, migration and invasion in pancreas cancer, while resistance to Src inhibition appears to be related to activation of parallel signaling pathways including STAT3. Furthermore, dasatinib inhibits the metastatic potential of pancreas cancer by inhibiting cell migration, invasion and anchorage-independent growth. The in vivo effects of dasatinib on pancreas tumor growth correlate with significant inhibition of Src and AKT phosphorylation, demonstrating biological and translational relevance.