Recent years have seen the emergence of therapeutics directed against specific signaling pathways critical for the onset and progression of cancer. Protein tyrosine kinases (PTKs), by the virtue of their regulation of cellular functions that contribute to cancer, including cell proliferation, survival, apoptosis, migration, and DNA damage repair, have emerged as new anticancer targets. Rational targeting of PTK activity to control these signaling pathways, and thus correct aberrant cellular behaviors in cancer, has been successful in improving outcomes of many types of cancer [1
]. Moreover, the specificity of these targeted drugs results in fewer and less severe side effects compared to conventional cancer treatments which are non specific in their actions. Of the approximately 20 classes of PTKs, the epidermal growth factor receptor (EGFR) family, whose members include HER1 (EGFR), HER2/neu (ErbB2), HER3 (ErbB3), and Her4 (ErbB4) [2
], has been the most widely studied. While the EGFR signaling cascade is essential for homeostasis, dysregulation of EGFR kinase activity has been implicated in the oncogenic transformation of cells [3
]. EGFR overexpression, gene amplification, mutations, and increased kinase activity have been observed in many solid cancers of epithelial origin including breast, lung, head and neck, ovarian, bladder, and pancreatic cancers [2
Specifically, frequent abnormal amplification or activation of EGFR has been observed in non-small cell lung cancer (NSCLC). Two small molecule EGFR tyrosine kinase inhibitors (EGFR-TKI), gefitinib (Iressa, AstraZeneca International) and erlotinib (Tarveca, OSI Pharmaceuticals) have been evaluated in patients with NSCLC [6
]. These ATP competitive, reversible EGFR-TKIs have been effective only in a small subset of NSCLC patients bearing somatic mutations (deletions in exon 19 and the L858R mutation) in the kinase domain of EGFR [8
]. Nevertheless, patients initially responding to TKI therapy invariably develop resistance to these drugs, thereby limiting progression-free survival to approximately 9-13 months with a median survival of 2 years [9
]. In the past several years, studies underpinned the molecular mechanisms responsible for drug resistance including acquisition of secondary mutation in EGFR kinase domain (threonine to methionine mutation, T790M) and/or c-MET amplification [10
]. However, these constitute only ~50-70% of EGFR-TKI resistant cases, indicating mechanisms leading to resistance in the remaining cases are yet to be unraveled. Recent endeavors have identified that in addition to increased receptor internalization or altered EGFR trafficking [14
], epithelial to mesenchymal transition (EMT) can be related with acquisition of resistance towards EGFR TKIs [15
EMT, characterized by the loss of cell-cell junctions, repression of E-cadherin expression and gain of mesenchymal markers significantly contributes to cancer invasion and metastasis. Recent evidence indicates EMT induction in tumor cells can also lead to emergence and/or enrichment of cancer stem cells (CSCs) [19
]. CSCs, also known as tumor initiating cells or cancer stem like cells, refer to a minor subpopulation of cancer cells with properties similar to somatic stem cells including self-renewal and multi-lineage differentiation. Initially identified in acute myeloid leukemia, CSCs have later been found in various cancers including breast, lung, brain, pancreatic, and prostate cancer [20
]. By the virtue of altered cell cycle kinetics, increased DNA repair response, increased expression of antiapoptotic regulators as well as transporter proteins, CSCs are able to survive radiation or chemotherapeutic insults [28
]. Thus, these cells are more refractory to cytotoxic agents compared to the differentiated cancer cells which constitute the bulk of the tumor. In fact it is believed that CSCs contribute significantly to tumor relapse following chemo or radiotherapy.
Based on these observations, we speculated that CSC selection during prolonged exposure to EGFR TKIs may play a role in eventual progression of cancer after a period of successful response. Recent evidence shows existence of a population of cells expressing cancer stem cell markers CD44high
in erlotinib resistant non small cell lung cancer (NSCLC) cell lines [15
]. However, to the best our knowledge these cells were not characterized in terms of their potential to self-renew, differentiate or induce resistance to EGFR-TKI therapy. In this study we generated an erlotinib resistant subline (H1650-ER1) from erlotinib sensitive lung cancer cell line NCI-H1650. Enrichment of cells with CSC markers and phenotypes in the resistant subline was confirmed by several techniques: (a) expression profiling of cell surface markers, (b) side population (SP) analysis (identification of a population of cells, called SP, characterized by high efflux of DNA-binding dye, Hoechst 33342 or DyeCycle Violet (DCV) dye by ABCG2, an ATP binding cassette transporter [29
]) and (c) culture of cells in suspension in serum free medium to promote generation of tumor spheroids.
Our studies demonstrate that the erlotinib resistant subline was composed of an increased population of cancer stem cell-like cells and exhibited enhanced colony formation ability in soft agar. SP cells isolated from H1650-ER1 showed self-renewal as well as differentiation potential. Furthermore, SP cells were more resistant to EGFR-TKIs than non-SP cells. These observations indicate that resistance to molecular targeted therapy could arise from selection and enrichment of cancer stem cell-like cells, which are intrinsically resistant to erlotinib.