Involvement of autophagy in the response of tumor cells to PtdIns3K inhibitors

Abstract

The phosphoinositide 3-kinase (PI3K) pathway plays a crucial role in cell proliferation and survival and is frequently activated by genetic and epigenetic alterations in human cancer. An arsenal of pharmacological inhibitors of key signaling enzymes in this pathway, including class I_A PI3K isoforms, has been developed in the past decade and several compounds have entered clinical testing in cancer patients. The PIK3CA/p110α isoform is the most studied enzyme of the family and a validated cancer target. The induction of autophagy by PI3K pathway inhibitors has been documented in various cancers, although a clear picture about the significance of this phenomenon is still missing, especially in the in vivo situation. A better understanding of the contribution of autophagy to the action of PI3K inhibitors on tumors cells is important, since it may limit or enhance the action of these compounds, depending on the cellular context.

The PI3K signaling pathway is involved in the control of key cellular functions, such as cell growth and proliferation, survival, metabolism, and migration. This intracellular signaling pathway is frequently activated by genetic and epigenetic alterations in human cancer. The PI3K family comprises eight catalytic isoforms, which are subdivided into three classes. The class I_A PI3K isoform PIK3CA is a validated drug target in the field of oncology, in particular because activating mutations in PIK3CA are frequent in human cancer. Lung cancer is a major cause of death and is subdivided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The latter represents about 13–15% of all cases of lung cancer and is associated with an overall 5-year survival rate of < 5%. A number of molecular alterations involved in SCLC pathogenesis have been reported, including upregulation of anti-apoptotic BCL2 proteins, overexpression of MYC family oncogenes, as well as genetic abnormalities in the tumor suppressor genes TP53 and RB1. The BCL2 family of proteins plays a crucial role in the survival of SCLC cell lines in vitro and in vivo. Moreover, amplification and mutations of the PIK3CA gene were identified in SCLC. The class I_A PIK3CA and PIK3CB/p110β isoforms are overexpressed in SCLC cell lines, in addition to constitutive activation of the AKT-MTOR pathway. PI3K signaling is also involved in the survival and proliferation of SCLC. Therefore, targeting this pathway with selective pharmacological inhibitors may lead to the development of novel and more effective therapies for SCLC.

We have investigated the potential of targeting the catalytic class I_A PI3K isoforms in SCLC. Overexpression of the class I_A PI3K isoform PIK3CA and the anti-apoptotic protein BCL2 was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K PIK3CA with RNA interference (RNAi) or selective pharmacological inhibitors results in strongly impaired growth of SCLC cells in vitro and in vivo. Inhibition of PIK3CA also results in increased apoptosis and autophagy, which is accompanied by decreased activation of the MTOR pathway. Surprisingly, inhibition of autophagy with chloroquine rescues part of the cell death induced by PI3K PIK3CA inhibitors. The degree of rescue observed upon autophagy inhibition is comparable to the rescue observed when apoptosis is inhibited by using a pan-caspase inhibitor. In addition, the PIK3CA inhibitors induce autophagy in some SCLC cell lines where apoptosis is not observed. We next hypothesized that PIK3CA controls the expression of a selective subset of genes implicated in SCLC cell proliferation and/or survival. A comparative DNA microarray analysis of SCLC cell lines in which either PIK3CA or PIK3CB is selectively inhibited reveals that PIK3CA inhibition profoundly affects the balance of pro- and anti-apoptotic BCL2 family proteins. The NFKB transcriptional network was found to control BCL2 expression downstream of PIK3CA. The PIK3CA inhibitors induce increases in both SCLC apoptosis and autophagy, which is consistent with BCL2 family proteins being a target of PIK3CA. BCL2 family proteins are key regulators of both apoptosis and autophagy, and their reduced expression upon inhibition of the PIK3CA-NFKB pathway may play an essential role in the effects of the PIK3CA inhibitors in SCLC. Thus, the induction of autophagy by PIK3CA inhibitors reflects reduced BCL2 expression and inhibition of MTOR.

We have previously evaluated the MTOR inhibitor everolimus in SCLC and found that it is effective in a subset of cell lines characterized by constitutive activation of the AKT-MTOR pathway. Intriguingly, autophagy inhibition also partially rescues cell death induced by everolimus, confirming the results obtained with PIK3CA inhibitors. Also of note is that our previous work in neuroblastoma has shown that the class I_A PI3K isoform PIK3CD/p110δ contributes to cell proliferation and survival by controlling the activation of the MTOR pathway and the expression levels of anti-apoptotic BCL2 family proteins. Therefore, the relative importance of class I_A PI3K isoforms in selected cancer types may, in part, be attributed to differences in expression levels. However, the role of class I_A PI3K isoform in the regulation of BCL2 family expression may be a more general function, which has an impact upon the control of both autophagy and apoptosis.

Our results in SCLC are in contrast to those reported by others on the role of autophagy in the response to PI3K inhibitors. In glioma and pancreatic adenocarcinoma, for example, autophagy suppression was reported to enhance the efficacy of inhibitors of the PI3K-AKT-MTOR pathway. It should be noted, however, that different types of inhibitors were used in these studies (such as dual PI3K-MTOR inhibitors) so these contrasting results cannot yet be definitively attributed to the differences in cancer types studied. However, further work should aim to elucidate the impact of the cellular context on the role of autophagy in the action of PI3K inhibitors. It will also be essential to further study the role of autophagy in in vivo models of human cancers treated with PI3K inhibitors. Such work is of importance in order to make substantial progress in the development of personalized medicine approaches for cancer patients that involve PI3K inhibitors.

Wojtalla A, Fischer B, Kotelevets N, Mauri FA, Sobek J, Rehrauer H, et al. Targeting the Phosphoinositide 3-Kinase p110-α Isoform Impairs Cell Proliferation, Survival, and Tumor Growth in Small Cell Lung Cancer. Clin Cancer Res. 2013;19:96–105. doi: 10.1158/1078-0432.CCR-12-1138.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.