Colon cancer continues to be the second most common cancer-related death in the United States (Jemal et al., 2008
). The etiology of mCRC is a complex series of genetic events that are characterized by several alterations including p53, EGFR and SFK expression and mutations in KRAS (Fearon and Vogelstein, 1990
; Summy and Gallick, 2003
). The EGFR protein is expressed in ~ 85% of mCRC as measured by the specific binding of 125
I-EGFR to tumor plasma membrane preparations, Western blotting and immunohistochemistry (Normanno et al., 2003
). In addition, It is estimated that 30–40% of patients with CRC have a KRAS mutation (Bardelli and Siena, 2010
; Normanno et al., 2009
). Further, it has been demonstrated in several clinical trials that patients with mCRC and a KRAS mutation do not respond to cetuximab therapy (Benvenuti et al., 2007
; Cappuzzo et al., 2008
; De Roock et al., 2008
; Di Fiore et al., 2007
; Karapetis et al., 2008
; Khambata-Ford et al., 2007
; Lievre et al., 2008
). These trial results leave a large population of patients with mCRC that cannot benefit from cetuximab therapy. The data presented herein indicate that dasatinib can sensitize cetuximab resistant, KRAS mutant CRC tumors to cetuximab. Further, this combinatorial treatment was marked by downregulation of components of the MAPK, AKT/mTOR, β-catenin and STAT pathways.
We screened 16 CRC lines for EGFR and SFK expression, and KRAS or BRAF mutations and dependency on KRAS signaling (). Next we determined if these model systems mimic clinical findings in that KRAS mutant CRC lines would be resistant to cetuximab therapy. To test this hypothesis we treated all KRAS mutant lines in vitro
and challenged them with increasing concentrations of cetuximab (data not shown). The results of this indicated that KRAS mutant CRC lines showed a robust response to cetuximab on plastic plates and did not mimic what is seen in vivo
and the clinic. Therefore we performed a series of cell culture experiments using plastic plates, fibronectin, laminin, fibronectin/laminin or PDL/laminin coated plates. These experiments indicated that PDL/laminin plates could most closely mimic clinical findings showing that KRAS mutant CRC lines were resistant to cetuximab (). This finding suggests that the interaction between the extracellular matrix in vitro
, and most likely in vivo
, plays a critical role in KRAS mutant CRC response to EGFR targeting agents. Viloria-Petit and colleagues reported that cetuximab resistant lines established in vivo
, were sensitive to cetuximab in vitro
(with plastic plates) after establishment of cell lines taken from mouse xenografts (Viloria-Petit et al., 2001
). Collectively these findings underscore the importance of the experimental approach to study therapeutic targeting KRAS mutant CRC lines and indicate that factors in the cell’s environment are critical in the treatment of KRAS mutant CRC.
In three KRAS mutant lines were tested for their response to cetuximab, dasatinib or the combination. Each line was resistant to cetuximab and semi-responsive to dasatinib. However, the combination of the two molecular targeting agents led to decreased proliferative potential as compared to either agent alone (). We verified that the cetuximab and dasatinib could reduce the activity of their respective targets (). Although, the EGFR couples growth factor signaling to the RAS/RAF/MEK/ERK pathway, and mutations in KRAS uncouple this pathway from the receptor, the EGFR still plays a role in the activation of other key pathways such as the PI3K/AKT pathway, STATs pathway and the PLCγ/PKC pathways (Marmor et al., 2004
). These pathways may still be activated by the EGFR, even in the KRAS mutant setting. To determine the effects of co-inhibition of SFKs and the EGFR we used phospho-array analysis on the three KRAS mutant CRC lines treated with vehicle, dasatinib, cetuximab or the combination. The results of these experiments revealed common pathways inhibited by the combination of these two agents in mutant KRAS CRC lines. Firstly, in LS180 and HCT116 the β-catenin pathway appeared to be downregulated (). This was evident by the decrease in phosphorylation of GSK3α and GSK3β. Decreased activity in this enzyme results in decreased β-catenin phosphorylation (also noted in the phospho-array), thus allowing it to translocate to the nucleus and where it binds the Lef/Tcf transcription factors and activating target genes involved in cancer progression. Secondly, in LS180 and HCT116, downregulation of the AKT/mTOR/p70S6 Kinase pathway was noted. In both lines activating phosphorylation events on AKT were decreased. AKT, through a series of complex signal transduction cascades, leads to the activation of the mTOR1 complex (Engelman, 2009
). This serine-threonine kinase then phosphorylates p70 S6 kinase which leads to the increased translation of mRNAs that encode proteins for cell cycle regulators (MYC and cyclin D1) as well as ribosomal proteins and elongation factors involved in translation (reviewed in (Rini, 2008
)). Finally, in all three lines tested, the combination of dasatinib and cetuximab resulted in the downregulation two pathways involved in tumor proliferation: 1)
members of the STAT family and 2)
members of the MAPK signaling cascade. The STAT family is comprised of seven members, STAT1-4, STAT5a, STAT5b and STAT6. Binding of growth factors or cytokines to their receptors results in intrinsic kinase activity or recruitment of receptor-associated kinases (janus kinase (JAK) and SFKs). These phosphorylated receptors in turn phosphorylates STATs on key residues leading to their dimerization and translocation to the nucleus where they regulate genes involved in cell proliferation, apoptosis, and angiogenesis and tumor growth. In terms of the MAPK signaling pathway the combination of dasatinib and cetuximab impacted proteins within this cascade albeit at different levels of the pathway. At the terminal end of the classical RAS/RAF/MEK/ERK cascade sits two proteins the 90 kDa ribosomal S6 kinase (RSK1) and MSK1/2. RSKs are phosphorylated at the end of the classical where ERK phosphorylates RSK1 in the kinase activation loop (Richards et al., 1999
). Activation of RSK1 can lead to the phosphorylation of the pro-apoptotic protein BAD that, when phosphorylated, abrogate BAD’s pro-apoptotic function (Shimamura et al., 2000
). In addition, RSK1 can phosphorylates IkBa, the inhibitor of NF-kB, inducing its degradation and allowing its translocation and function in the nucleus (Ghoda et al., 1997
). Decreased RSK1 phosphorylation was noted in LS180 and HCT116. MSK1/2 are believed to play a pivotal role in the activation of the CREB transcription factor by phosphorylation of serine 133 (Wiggin et al., 2002
). This molecule along with MEK1/2 was down regulated in LoVo. Collectively these data suggest that therapeutic treatment with dasatinib and cetuximab results in the downregulation of several critical pathways involved in the progression of cancer.
Both in vitro
and in vivo
( and ) the HCT116 data demonstrate a statistically significant response to the combination of cetuximab and dasatinib, but not as robust as compared to LS180 or LoVo. This may be explained due to the reported PI3 kinase mutation in HCT116 (Jhawer et al., 2008
; Wee et al., 2009
), which would lead to enhanced signaling through the AKT pathway, independent of cetuximab treatment. However, AKT activity, as measured by phospho-array analysis () did show decreased AKT activity as compared to either agent alone. This suggests that other, yet to be identified mechanisms exist for the decreased response to the combination in the HCT116 cell line.
Dasatinib is an orally bioavailable and promising therapeutic agent for the treatment of several human malignancies including chronic myelogenous leukemia, non-small cell lung cancer, small cell lung cancer, advanced breast cancer (including triple negative), pancreatic cancer, prostate cancer and head and neck squamous cell carcinoma (reviewed in (Kim et al., 2009
). Dasatinib was discovered through the synthesis and testing of a series of thiazole-based compounds with activity against SRC and ABL kinases to target imatinib-resistant BCR-ABL mutants (Kantarjian et al., 2006
). Dasatinib, although relatively specific for ABL, BCR-ABL and the SFKs, it possesses a broad-spectrum of inhibition of kinases including Kit, PDGFR, EphA receptors and several others (Hantschel et al., 2008
). Non-specific effects must always be considered when developing a mechanism but regardless, the effect of cetuximab and dasatinib on anti-tumor growth is evident and dasatinib’s broad spectrum of kinase inhibition may, in part, be linked to its clinical success thus far as well as in combination with cetuximab in the KRAS mutant CRC setting. The combination of cetuximab and dasatinib has shown to be effective in other circumstances these include in the situation of overcoming acquired resistance to cetuximab in NSCLC (Li et al., 2009
; Wheeler et al., 2009
). In addition, clinical trials looking at this combination are currently in recruitment in HNSCC, mCRC and other solid tumors (clinical trials.gov).
KRAS is clearly a marker of resistance to cetuximab in monotherapy for CRC and patient screening is still essential. However, our results suggest KRAS mutant CRC lines are dependent on both signals from the EGFR and SFKs. Thus, the relationship between EGFR and SFK signaling in the presence of KRAS mutations will be an area of intense investigation. The concomitant treatment of dasatinib and cetuximab may be a viable option for KRAS mutant CRC patients without PI3K, or further downstream mutations. In addition, future directions may include investigations of this combination in the KRAS wild type setting. In summary, this study combines two FDA-approved agents, dasatinib and cetuximab, in the KRAS mutant CRC setting. From the data provided it appears that dasatinib can sensitize KRAS mutant tumors to cetuximab. This work may provide rationale for further investigative clinical trials using dasatinib plus cetuximab in patients with KRAS-mutant, cetuximab-resistant mCRC.