Suppression of Stat3 activity sensitizes gefitinib-resistant non small cell lung cancer cells

Abstract

Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a small subset of non small cell lung cancer (NSCLC) harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR inhibitors, suggesting that other factors are implicated in survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) function as transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been discovered in a number of cancers. In this study, we found that a small molecule, reactivation of p53 and induction of tumor cell apoptosis (RITA), showed anti-cancer activity against gefitinib-resistant H1650 cells through a p53-independent pathway. Stat3 suppression by RITA attracted our attention to investigate the role of Stat3 in sustaining survival of H1650 cells. Pharmacological and genetic approaches were employed to down-regulate Stat3 in H1650 cells. WP1066, a known Stat3 inhibitor, was shown to exhibit inhibitory effect on the growth of H1650 cells. Meanwhile, apoptosis activation by siRNA-mediated down-regulation of Stat3 in H1650 cells provides more direct evidence for the involvement of Stat3 in viability maintenance of H1650 cells. Moreover, as a novel identified Stat3 inhibitor, RITA increased doxorubicin sensitivity of H1650 cells in vitro and in vivo, suggesting that doxorubicin accompanied with Stat3 inhibitors may be considered as an alternative strategy to treat NSCLC patients who have inherent resistance to doxorubicin. Overall, our observations reveal that targeting Stat3 may be an effective treatment for certain NSCLC cells with oncogenic addition to Stat3.

Introduction

Lung cancer is the leading cause of cancer-related death worldwide. More effective therapies are needed due to the poor survival rates in patients of lung cancer. Lung cancer is broadly classified into two major categories: small cell lung cancer (SCLC) and NSCLC. Clinical reports have shown that 80% of lung cancers are diagnosed with NSCLC [1]. Platinum-based chemotherapy has become the standard treatment for NSCLC based on the results from numbers of clinical trials [2], [3], [4]. Nowadays, target therapies causing no or minor side effects may compensate the incompleteness of conventional chemotherapies. A strong correlation is revealed between the expression levels of ErbB family and the malignant proliferation [5]. EGFR mutations are identified in 10–15% of Caucasian patients with higher percentage in Asian patients and can result in lung cancer pathogenesis [6]. EGFR has thus become a promising therapeutic target for the treatment of NSCLC.

EGFR is a member of the ErbB family that is classified as receptor tyrosine kinase (RTK). Activated EGFR phosphorylates downstream targets, including phosphoinositide 3-kinase (PI-3K), phospholipase C-γ (PLC-γ), extracellular signal regulated kinase (Erk), and Stat3/Stat5 to promote cell proliferation and survival. The majority of NSCLC-associated mutations occur as an in-frame deletion of exon 19 (DelE746-A750) or a point mutation in exon 21 (L858R), leading to constitutively activated EGFR [7]. Patients with certain EGFR mutations have a higher response rate to an EGFR targeted drug, gefitinib (ZD1839, Iressa), than those with wild type EGFR [8], [9], [10], [11]. However, carrying EGFR mutations does not assure NSCLC patients of the sensitivity to EGFR inhibitors [12], [13], [14]. Similar outcome was observed in cell-based studies as well. H1650, a NSCLC cell line carrying mutant EGFR (DelE746-A750), exhibits much greater resistance to gefitinib compared to other cell lines which carry drug-sensitive mutations within EGFR [15]. Thus, it is important to develop more effective therapeutic strategies to treat NSCLC with the resistance to current therapies.

Stat3, a member of STAT family, is activated mainly through the phosphorylation at Tyr705 by receptor or non-receptor tyrosine kinases including EGFR, Janus activated kinase (JAK), and c-Src [16]. Following activation, Stat3 dimerizes via its SH2 domain, translocates to the nucleus, and functions as a transcription factor to induce the expression of target genes. Stat3 activation is accompanied by the upregulation of cyclin D1, c-Myc, and Bcl-X_L to promote cell survival and proliferation [17]. Dysregulation of Stat3 activity is associated with the pathogenesis of numerous types of cancers including breast, colon, cervical, and prostate cancers [18]. Several lines of evidence have also indicated the correlation between Stat3 activity and pathogenesis of lung cancer [19], [20].

Due to the strong correlation between Stat3 activation and tumorigenesis, suppression of Stat3 by genetic or pharmaceutical modalities has been shown to have anti-tumor effects in vivo and in vitro [21]. Phosphotyrosyl peptides with the Stat3 SH2 domain-binding activity were shown to inhibit Stat3 activation [22]. Gao et al. demonstrated the effectiveness of vector based RNA interference to suppress Stat3 and its downstream effectors [23]. In their study, inhibition of Stat3 activity using the same approach reduces the survival of PC3, a prostate cell line, in vivo and in vitro. Recently, small molecule inhibitors, including WP1066, FLLL31, and FLLL32, for upstream effectors of Stat3 have also shown their anti-tumor activity in pancreatic, breast, and malignant glioma cancer cells [24], [25]. These observations strongly suggest that Stat3 may be a promising molecular target for drug development.

Doxorubicin is widely employed to treat a variety of cancers such as lymphoma, leukemia, breast, lung, ovarian, gastric, and thyroid malignancies [26]. Doxorubicin induces DNA damage through the generation of free radicals and inhibits topoisomerase II in cancer cells [27]. Although doxorubicin is very potent to treat cancers, resistance to doxorubicin and its toxicity, e.g., heart damage, have restricted its applications. Once a cumulative dose of doxorubicin exceeds the safety threshold, doxorubicin is generally excluded from the regimen to reduce the incidence of doxorubicin-induced cardiotoxicity [28]. Unfortunately, this implies that patients may be forced to give up such an effective therapeutic strategy to avoid the severe side effects. Thus, it will be important to assess whether the chemosensitivity to doxorubicin in cancers could be enhanced by combination with other agents.

In this study, we found that RITA could sensitize gefitinib-resistant H1650 cells probably through its suppression of Stat3 activity. The role of Stat3 in maintaining the survival of H1650 cells was further evaluated using WP1066, a known Stat3 inhibitor, and small interfering RNA (siRNA) technology. Results from the pharmacological and genetic characterizations indicate that Stat3 is essential to maintain cell viability of H1650 cells. Moreover, the functional significance of RITA-mediated Stat3 inhibition in H1650 cells was determined by examining its effects on the sensitivity of H1650 cells to doxorubicin, a relatively ineffective agent for NSCLC [29], [30]. Altogether, our studies suggest that the development of Stat3 targeting drugs may be beneficial for NSCLC patients who do not respond to current therapeutics.