Autophagy was designated as programmed cell death type II, whereas apoptosis is well-known as programmed cell death type I. However, recently autophagy was found to promote cellular survival under unfavorable conditions such as deprivation of amino acids or ATPs, revealing a new role of autophagy in cancer development.
Autophagy is morphologically characterized by the appearance of “double-membrane” vacuoles (autophagosomes) in the cytoplasm. In addition, the mammalian homologue of the yeast protein Apg8p, (also named LC3), is found to be a specific biochemical marker for autophagy. Newly synthesized LC3 termed LC3-1 is evenly distributed throughout the cytoplasm. Upon induction of autophagy, some LC3-I is converted into LC3-II, which is tightly bound to the autophagosomal membranes forming ring-shaped structures in the cytosol. We confirmed biochemically and morphologically that autophagy can be activated by gefitinib or erlotinib, two well-used EGFR-TKIs, in two independent lung cancer cell lines. Interestingly, a very recent paper reported that autophagy can be induced by anti-EGFR antibodies 
. Both EGFR antibodies and EGFR-TKIs are widely used to treat cancer patient by inhibition of EGFR, revealing a new link between EGFR inhibition and autophagy activation.
Autophagy can be activated as the cellular response to cancer therapy. A number of cancer therapeutics including DNA-damaging chemotherapeutics, endocrine therapies (e.g. tamoxifen) and radiation therapy have been found to induce autophagy in vitro and in vivo 
. Recently, it was found that autophagy can be activated and protected tumor cells from targeted therapies, such as the imatinib mesylate in philadelphia chromosome–positive cells 
, trastuzumab in breast cancer 
, Src family kinase inhibitors in prostate cancer 
, proteasome inhibitors in prostate cancer 
. Consistently, we found that autophagy can be activated by gefitinib and erlotinib in lung cancer and promote cellular survival in the target therapy using EGFR-TKIs. Blockage of autophagy by pharmacological or genetic approaches greatly enhanced the growth inhibitory effect of gefitinib or erlotinib. Thus, inhibition of autophagy has the potential to improve the clinical efficacy of EGFR-TKIs for cancer treatment.
As a sensor of amino acids and ATP, mTOR negatively regulates autophagy. Indeed, we found that both TKIs can inhibit the activation of mTOR as well its upstream regulator, PI3K/Akt. Another signaling pathway important to autophagy is Raf/MAPK pathway 
. However, we failed to find a clear correlation between Raf/MAPK pathway and autophagy in cell lines we used. This is probably due to oncogenic mutations predominantly occurred in Ras/MAPK pathways. For instance, k-Ras gene was known to be mutated in A549 cells. Clinically, patients with k-Ras mutations failed to response to TKIs treatment. It would be interesting to know whether patients with k-Ras mutations could benefit from the combination of TKIs and inhibitors of autophagy.
Interestingly, our results indicated that gefitinib or erlotinib induced autophagy might be EGFR independent. As shown in , both EGFR-TKIs could induce autophagy even EGFR expression was greatly reduced. Although gefitinib and erlotinib were developed as the specific inhibitors targeted to the kinase domain of EGFR, however, recent results indicted that these two inhibitors can have other targets, such as non-receptor tyrosine kinases that acts also upstream of PI3K/Akt/mTOR pathway 
. Consistently, large scale clinical trials confirmed that measurement of EGFR expression by immunohistochemistry was not useful for picking up patients to be benefited from gefitinib therapy. Certainly, we still could not completely exclude the relevance of EGFR in gefitinib or erlotinib induced autophagy since certain amount of EGFR was still detected after knockdown by EGFR siRNAs. Indeed, EGFR-TKIs activated autophagy was much more enhanced by siRNA 1 which displayed better knockdown efficiency than siRNA 2 (). Hence, we need further investigations to clarify the role of EGFR in the gefitinib or erlotinib-induced autophagy. Nevertheless, EGFR-TKIs and EGFR siRNA had a synergetic effect on the induction of autophagy, which could be specifically inhibited to increase the clinical efficacy of targeted therapy.
In summary, our work reinforced the notion that cancer cells can survive in a stressful environment, following inhibition of critical oncogenic pathways, by inducing autophagy. These tumor cells are primed to resume proliferation once drug concentration drops after drug withdrawal due to toxicity or mutations develop to confer drug resistance. Thus, in combination of therapeutic strategies that aim to inhibit autophagy in patients treated with conventional chemotherapy or novel targeted therapy with EGFR-TKIs represents a promising approach with higher efficacy for cancer patients.