Treatment for pancreatic cancer remains a therapeutic challenge due to the lack of any effective therapy. Despite a greater understanding of the molecular pathways involved in pancreatic tumorigenesis, the use of individual targeted agents, have failed to provide meaningful improvements in survival. We investigated a strategy of targeting multiple tumor-associated pathways, namely, activated EGFR and Src tyrosine kinase signaling, which act as complementary pathways in pancreatic cancer to promote tumor growth. Nine human pancreatic cancer cell lines were characterized for their expression of Src and EGFR, phosphorylated Src and EGFR () and sensitivity to dasatinib, erlotinib and gemcitabine, identifying cell lines that were sensitive and resistant to these agents (). Interestingly, pancreatic cancer cell lines that were sensitive to dasatinib were also sensitive to erlotinib and gemcitabine treatment, while cell lines that were more resistant to dasatinib also showed greater resistance to erlotinib and gemcitabine therapy, suggesting an inherent resistance to individual cytotoxic or targeted therapies.
Numerous reasons exist to expect that targeting multiple tumor-associated pathways will enhance the therapeutic effects of cancer treatment. As stated above, even though the genetic alterations associated with pancreatic cancer can be associated with a core set of 12 signaling pathways, the processes that are genetically altered vary amongst each individual patient (2
). In addition, the interplay between tumor cells and surrounding supportive cells such as vascular endothelial cells and pericytes, fibroblasts and immune cells adds to the complexity of altered cellular signaling to stimulate tumor growth, clearly suggesting that targeting a single component will not result in sustained inhibition of tumor growth. Moreover, even with the use of cytotoxic or targeted therapies, parallel and reciprocal signaling pathway activation can promote resistant cell clones to continue to expand, resulting in refractory tumor response to a given agent that affects a single mechanism of action. Therefore, targeting multiple signaling pathways involved in tumor growth has the potential to overcome either primary or acquired resistance to targeted monotherapy and increase the likelihood of sustained response by affecting different mechanisms of action associated with cancer development and enhance the effects of conventional cytotoxic chemotherapy. Our results clearly indicate that combined inhibition of Src and EGFR signaling results in improved inhibition of pancreatic tumorigenesis and potentiates the effect of gemcitabine chemotherapy, the addition of which further enhances the anti-tumor effects of EGFR and Src inhibition.
Resistance to cytotoxic chemotherapy has remained a major factor in the poor outcomes seen in patients with pancreatic cancer. Inhibition of Src signaling has been shown to restore sensitivity to gemcitabine and 5-flourouracil in human pancreatic cancer cells (19
). Similarly, silencing of FAK expression in PANC1 cells restores sensitivity to gemcitabine treatment (27
). In addition, Src inhibition has also been shown to increase sensitivity to other cytotoxic agents such as oxaliplatin in colon cancer (28
). This implicates Src inhibition in restoring chemosensitivity and suggests a novel approach using dasatinib in combination with gemcitabine for the treatment of pancreatic cancer. However, we show that at IC50
doses of dasatinib, erlotinib and gemcitabine as individual agents or as double combinations of agents have minimal effects on inhibiting signaling of multiple tumor-associated pathways in both BxPC3 and PANC1 cells, whereas only the triple combination dasatinib and erlotinib with gemcitabine resulted in cooperative inhibition of these pathways (). These results support the idea that modulation of chemoresistance may require the inhibition of both Src and EGFR signaling, requiring targeting of different pathways at multiple levels.
The heterogeneity of genetic alterations associated with pancreatic cancer, as with most solid tumors for that matter, makes it highly unlikely that a “one size fits all” approach will be effective and the need to identify biological markers that can predict treatment response (or lack of response) will be essential to optimize targeted treatment strategies. We have previously shown that activated STAT3 is a biomarker of resistance to Src inhibition with dasatinib in resistant PANC1 cells (14
). STAT3 is activated by a variety of growth factors receptors such as EGFR via non-receptor tyrosine kinases such as Src family kinases (SFKs) (29
). STAT3 activation regulates oncogenic signaling in many tumor types and leads to increased cell survival, proliferation and tumor growth (30
). Constitutively active STAT3 is sensitive to both EGFR and Src inhibition (31
). However, despite early suppression of aberrant STAT3 signaling, sustained EGFR and Src inhibition can result in Janus kinase (JAK)-mediated reciprocal activation of STAT3 signaling (30
). This type of feedback and parallel signaling limits the effectiveness of individual targeted therapies as seen in our results in which dasatinib or erlotinib, either individually or in combination did not result in inhibition of activated STAT3 signaling. Only the combination of dasatinib and erlotinib with gemcitabine overcame STAT3-mediated resistance of EGFR and Src inhibition both in vitro
and in vivo
. These results implicate treatment strategies not only attacking multiple targets, but combining these multi-targeted approaches with cytotoxic chemotherapies.
Although our results show that this triple combination of agents optimizes pancreatic cancer therapy, the addition of multiple targeted or cytotoxic drugs can also add potentially toxicity when administered to patients in combination. This emphasizes the importance of identifying molecular markers of resistance, such as activated STAT3, to tailor therapies targeting specific resistant pathways. We confirmed the role of activated STAT3 as a biomarker of resistance to Src inhibition.
Our results clearly suggest that the combination of dasatinib and erlotinib with gemcitabine may be a potent treatment regimen for pancreatic cancer and overcomes STAT3 mediated resistance of inhibition of pancreatic tumorigenesis. Taken together, our findings provide compelling evidence establishing the role of combined targeted therapy with cytotoxic chemotherapy as a paradigm to overcome resistance associated with reciprocal and parallel signaling seen with biologically targeted monotherapy. In addition, identifying biomarkers of resistance to targeted therapy, such as activated STAT3 signaling, can result in tailoring treatment to target specific resistant pathways, thereby limiting the toxicity associated with delivery of multiple agents.
The failure of conventional chemotherapeutic regimes to produce any meaningful impact on survival in patients with pancreatic cancer highlights a desperate need for novel treatment strategies. Src inhibitors represent a novel class of targeted drugs that have disease activity in several tumor types, however, have shown limited single-agent activity. One of the mechanisms of resistance to Src inhibition appears to be related to a lack of inhibition of activated STAT3 signaling. This study characterizes the in vitro and in vivo molecular effects of targeting two complementary tyrosine kinase pathways, Src with dasatinib and EGFR with erlotinib, combined with gemcitabine chemotherapy. Our results suggest that this combination of agents may be a potent treatment regimen for pancreatic cancer and provide evidence that combined targeted biological therapy in addition to cytotoxic chemotherapy can overcome treatment resistance. Such treatment strategies may be used to tailor therapy based on identified biomarkers of resistance to targeted monotherapy.