The rationale for using EGFR-targeted approaches for cancer treatment is now firmly established and numerous clinical trials are in progress. However, resistance to EGFR-targeted therapy universally emerges over time. The next challenge is to develop novel treatment strategies to overcome the intrinsic and acquired resistance. Some natural products such as the food derivatives I3C and DIM have antitumor properties and can prevent carcinogen-induced tumorigenesis and human tumor cell xenograft development in animal models as well as inhibit cancer cell growth in ErbB-receptor expressing cancer cells [12
]. We investigated the inhibitory effects of DIM on human cancer cells of the breast, lung, and central nervous system (glioma) which express mutant forms of the EGFR. Our results report, for the first time, that although the breast and glioma cells expressing EGFRvIII or non-small cell lung cancer cells expressing EGFR mutations can lead to resistance to targeted therapies, and in some cases, chemotherapy or radiation, these cells are sensitive to the growth inhibition induced by DIM.
EGFR mutants can heterodimerize with other oncoproteins and crosstalk with other receptor tyrosine kinases and activate multiple downstream signaling networks. This crosstalk can occur either through a direct association between receptors, or indirectly, via common interaction partners or downstream signaling molecules. This multi-layered crosstalk can result in the induction of resistance to targeted therapies. In breast cancer, EGFRvIII is frequently co-expressed with ErbB2, and defective degradation pathways of EGFRvIII can lead to prolonged and enhanced EGFRvIII/ErbB2 signaling [47
]. Although EGFRvIII exhibits defective degradation, we found that EGFRvIII was significantly down-regulated in breast cancer cells by DIM. Furthermore, high ErbB2-expressing, estrogen-independent, tamoxifen-resistant MDA-MB-361 breast cancer cells expressing EGFRvIII were much more sensitive to the growth inhibitory effects of DIM in comparison to low ErbB2-expressing, estrogen-dependent, tamoxifen-sensitive MCF-7 breast cancer cells expressing EGFRvIII [35
]. The reduction of EGFRvIII and ErbB2 expression in MCF-7 breast cancer cells was also much less pronounced than that of in MDA-MB-361 breast cancer cells expressing EGFRvIII, suggesting that DIM treatment may overcome EGFRvIII-mediated tamoxifen resistance in breast cancer. Targeting the expression of EGFRvIII and ErbB2 protein, but not its activity may be of clinical benefit to breast cancer patients expressing EGFRvIII.
The expression of Met has been implicated in the progression of a wide variety of cancers, including glioma, lung, and breast cancer [36
]. Amplification of Met and HGF has been associated with highly invasive and metastatic tumors and poor patient prognosis in lung cancer and amplification of Met has recently been linked to TKI-resistant lung cancer [39
]. Furthermore, the activity and expression of Met has been suggested to crosstalk with EGFR and synergize with EGFRvIII [5
]. Our study demonstrates, for the first time, that DIM can simultaneously reduce the expression levels of Met and EGFR mutants in glioma and lung cancer cells. Hence, as a single agent, DIM can eliminate the oncogenes that drive tumorigenesis, and DIM is also able to prevent “oncogene switching” by targeting various oncogenic receptor tyrosine kinases simultaneously. Moreover, DIM may circumvent TKI resistance in EGFR mutant-expressing non-small cell lung cancer cells and radiation resistance in EGFRvIII-expressing glioma cells. These results suggest that DIM may be used in combination with conventional therapeutics for the treatment of human malignancies expressing EGFR mutants.
The complexity of signaling networks that develop in cancer cells frequently results in a redundancy and overlap of cell survival and proliferation pathways, potentially allowing cancer cells to circumvent the therapeutic effects of targeting one single growth or survival signaling pathway. Since the ErbB-receptor signaling network is enormously diversified, cancer cells exhibit deregulation in multiple cellular signaling pathways. It was of no surprise that signaling cascades downstream of growth factor receptors were inhibited by DIM. Down-regulation of EGFR mutant activity and protein levels by DIM resulted in diminished Akt activity, enhanced expression levels of p21 or p27, and reduction of CDK4 and CDK6, which subsequently attenuates the progression from the G1 to S phase of the cell cycle. DIM treatment also induced PARP1 cleavage, reduced the expression of Surviving, and modulated the apoptotic pathway to achieve the inhibition of cancer cell proliferation.
Suppression of the proliferative p44/42 MAPK pathway by DIM treatment was also observed in all the tested cell lines, with the exception of the H4 glioma cells with and without EGFRvIII expression, which had an increased activation of p44/42 MAPK activity. This increased p44/42 MAPK activity in H4 glioma cells resulted in an up-regulation of Cyclin D1. Although this result is counter-intuitive, it has been shown that the blockade of the Akt pathway with pharmacologic agents often results in increased activation of the p44/42 MAPK pathway [51
]. This feed-back mechanism may be a characteristic of cancer cells which have the ability to activate different signaling pathways in order to have a survival or growth advantage under stress conditions. Nevertheless, DIM-mediated down-regulation of the Akt pathway was able to alter the expression of cell cycle regulators and induce apoptotic changes to overcome this feedback regulation. Furthermore, early studies showed that DIM treatment induces activation of the p38 MAPK pathway, leading to apoptosis of cancer cells [29
]. This stress activated pathway may play a significant role in DIM-mediated inhibition of ErbB-receptor expression as activation of this pathway has been shown to induce internalization and degradation of EGFR [52
]. Activation of p38 MAPK pathway may also play a role in DIM-mediated down-regulation of EGFR mutants as activation of p38 MAPK upon DIM treatments was clearly evident in these drug-resistant EGFR mutant-expressing cancer cell lines.
Taken together, we demonstrated that DIM can down-regulate multiple oncogenes and block multiple important signaling pathways that are responsible for promoting cancer cell survival and growth. Most importantly, our findings suggest significant potential clinical benefits as previous results have shown that EGFRvIII expression in cancer cells not only increases the invasiveness of cancer cells, it also renders these cells resistant to conventional cancer treatments such as chemotherapy and radiation. Non-small cell lung cancer cells also expressing EGFR mutations (T790M) are also resistant to TKIs, but DIM may be used as an alternative to inhibit the growth of these tumors.
An additional clinical benefit may be the prevention of metastatic disease or recurrence as DIM has the ability to decrease the invasive potential of aggressive cancer cells expressing EGFR mutants. The mechanism by which DIM inhibits cancer cell migration/invasion is largely unknown, although a few reports suggest the DIM suppresses the expression of the pro-metastatic chemokine receptor CXCR4 and its ligand CXCL12 [54
]. Therefore, non-toxic natural products from natural resources could be useful in combination with conventional chemotherapeutic agents for the treatment of drug-resistant human cancers with lower toxicity and higher efficacy. Our future studies will address the mechanism by which DIM inhibits the enhanced invasive potential mediated by EGFRvIII in cancer cells and whether DIM alone or in combination with other targeted therapies can inhibit increased tumorigenesis associated with cancer cells expressing EGFR mutants.