Although EGFR is activated through ligand binding and autophosphorylation of its cytoplasmic tail, it is well established that Src, or Src family kinases (SFKs), are necessary for full activation of the EGFR (Biscardi et al., 1999a). Src is the prototype member of a family of non-receptor tyrosine kinases (nRTKs) including Src, Yes, Fyn, Lyn, Lck, Hck Fgr, Blk and Yrk. These cytoplasmic membrane associated nRTKs are transducers of mitogenic signaling emanating from a number of RTKs including EGFR, HER2, fibroblast growth factor receptor (FGFR), platelet derived growth factor (PDFGR), colony-stimulating factor-1 receptor (CSF-1R) and hepatocyte growth receptor (Belsches et al., 1997; Bowman et al., 2001; DeMali et al., 1999; Mao et al., 1997; Muthuswamy et al., 1994; Tice et al., 1999). Investigations into the molecular interactions between SFKs and EGFR have revealed that SFKs can physically associate with activated EGFR (Belsches-Jablonski et al., 2001; Maa et al., 1995; Muthuswamy and Muller, 1995). This interaction results in a conformational change in the SFK and leads to autophophorylation at Y419 and transient activity (Xu et al., 1999). This interaction of SFKs with RTKs can result in enhanced or synergistic SFK activation and has been demonstrated in tumor types, most notably in HNSCC, NSCLC and CRC (Fu et al., 2008; Koppikar et al., 2008; Mao et al., 1997; Zhang et al., 2007).

Activation of SFKs occurs with high frequency during the development of CRC. An increase in SFK activity in CRC tumors as compared to normal adjacent mucosa has been reported (Bolen et al., 1987; Cartwright et al., 1989). In addition, activation of SFKs was reported at an early stage of colorectal tumor development in polyps with high malignant potential but not in small benign polyps of the colon (Cartwright et al., 1990). Further, premalignant ulcerative colitis epithelium has been reported to have elevated SFK activity (Cartwright et al., 1994), suggesting that SFKs activity may be a critical step in the development from non-malignant to malignant transformation in CRC. Talamonti et al reported increased activity and expression of SFKs in progressive stages of human colorectal cancer, suggesting that colon cancer progression may be dependent on increased SFK protein level and subsequent activity (Talamonti et al., 1993). Similar studies by Termuhlen et al looking at colorectal metastases to either the liver or the regional lymph nodes exhibited increased SFK activity levels when compared to the primary tumor (Termuhlen et al., 1993). Collectively these studies suggest a putative link between increased SFK activity and metastatic potential. Irby et al indicated that overexpression of normal c-Src in poorly metastatic human colon cancer cells enhances primary tumor growth but not the metastatic potential of these cancers (Irby et al., 1997). Additional studies by Irby et al cited that activating mutations in Src, as compared to increased expression and activity of Src, in a subset of human colorectal cancers might have a role in the malignant progression of human CRC (Irby et al., 1999). It has been reported that increased SFK expression occurs in approximately 80% of CRC specimens when compared to the normal adjacent colonic epithelium (Hurwitz et al., 2004). Recent studies looking at 64 individual CRC cell lines found a striking diversity of SFK activity. The authors reported that all lines tested depended on SFK activity for growth and concluded from this work SFK activity is important for the growth of CRC lines (Emaduddin et al., 2008). In addition to SFK activity and CRC progression, SFK activity has been reported as a marker of poor clinical prognosis (Aligayer et al., 2002). Collectively these investigations provide a wide body of evidence implicating Src and its family of kinases in CRC development and progression (for further review (Summy and Gallick, 2003)). Regardless of the form of activation, activated SFKs lead to the phosphorylation of several targets including the EGFR, STATS, PLCγ, PKCs, FAK, RAS, RAF and mucin 1 (MUC1) (Biscardi et al., 2000; Bivona et al., 2003; Blake et al., 1999; Bromberg et al., 1998; Chiu et al., 2002; Denning et al., 1996; Fabian et al., 1993; Joseloff et al., 2002; Kijima et al., 2002; Kronfeld et al., 2000; Li et al., 1994; Li and Kufe, 2001; Li et al., 2001a; Li et al., 2001b; McLean et al., 2005; Schaller et al., 1994; Yu et al., 1995).

Targeting EGFR has been intensely pursued in the last decade and has resulted in the FDA approval of five new molecular targeting agents since 2003 in four distinct solid tumors including metastatic, NSCLC, HNSCC, breast cancer and colorectal cancer (mCRC). One molecular strategy of EGFR inhibition has been the development of monoclonal antibodies (mAb, cetuximab and panitumumab) directed against the extracellular domain of the EGFR. This approach results in 1) blockade of endogenous ligand binding to the receptor, 2) inhibition of dimerization with other HER family members and 3) receptor internalization and degradation. Cetuximab and panitumumab have been approved for the treatment of mCRC when used alone or in combination with irinotecan in patients with irinotecan-refractory mCRC (cetuximab) or as a single agent in patients who do not respond to all available chemotherapies (panitumumab).

Despite the approval of this promising biological therapeutics many individuals do not respond to this class of drug. Intensive clinical trials have evaluated the outcomes of patients with mCRC in relation to their KRAS mutational status. The conclusions of this analysis demonstrated a strong correlation between mutated KRAS and a lack of response to cetuximab therapy indicating that KRAS status as a predictive factor (Amado et al., 2008; Bokemeyer 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; Punt et al., 2008; Van Cutsem et al., 2008). One the basis of these clinical trials ASCO published guidelines that strongly support the use of anti-EGFR antibodies in mCRC patients with wild type KRAS status (Allegra et al., 2009; Bardelli and Siena, 2010). These guidelines leave very few therapeutic options for mCRC patients harboring a KRAS mutation.

We screened 16 CRC lines for EGFR and SFK expression, and KRAS or BRAF mutations and dependency on KRAS signaling (Figure 1). 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 (Figure 2A). 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 figure 2B and 2C 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 (Figure 2B). We verified that the cetuximab and dasatinib could reduce the activity of their respective targets (Figure 2C). 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 (Figure 3A and 3C). 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 (Figures 2B and ​and5C)5C) 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 (Figure 3C) 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).