Closing escape routes: inhibition of IL-8 signaling enhances the anti-tumor efficacy of PI3K inhibitors

The phosphoinositide 3-kinase (PI3K) pathway serves as a relay where signals that emanate from the cell membrane are received and converted into intracellular signals that promote proliferation and survival. Inhibitors of PI3K hold promise for the treatment of breast cancer because activation of this pathway is highly prevalent. However, as is increasingly observed with inhibitors of cell signaling, there appear to be mechanisms of primary and secondary resistance. Britschgi and colleagues report that compensatory activation of the IL-8 signaling axis is a mechanism of primary resistance to PI3K inhibitors in some triple-negative breast cancers. In a set of experiments that carefully emulate the clinical scenario in a mouse model, they show that simultaneous inhibition of Janus kinase 2 enhances the efficacy of PI3K/mammalian target of rapamycin inhibition. Their paper lends further support to the concept that successful design of treatments with signal transduction inhibitors must anticipate potential escape routes - and include agents to simultaneously block them.

Metastatic triple-negative breast cancer (TNBC) remains an unsolved clinical problem. Patients are typically treated with cytotoxic chemotherapy that severely impedes their quality of life, response rates decline with each subsequent treatment regimen, and even when remission is achieved it is temporary and tumor progression occurs within a few months. Th e promise of targeted treatments is that, because of their specifi city for the tumor cell's signaling machinery, they could potentially suppress tumor growth for a prolonged period and at lesser cost to quality of life. Two key challenges in the design of such studies are fi nding the right target and anticipating and counteracting resistance mechanisms.
Th ere is a good rationale to target phosphoinositide 3-kinase (PI3K) in breast cancer, including TNBC: 30 to 40% of estrogen receptor-positive breast cancer, 20 to 30% of Her2-amplifi ed breast cancer, and 7 to 20% of TNBC have activating mutations of PIK3CA (encoding the p110α subunit of PI3K) [1][2][3][4]. While the frequency of activating mutations in PIK3CA is relatively low in TNBC, an increase in epidermal growth factor receptor expression [5,6] and inactivation of the inhibi tory phospha tases PTEN and INPP4B [7,8] are frequent, and thus activation of the PI3K pathway is also highly prevalent in TNBC.
Th ese fi ndings have led to a number of preclinical and now ongoing clinical studies examining the effi cacy of PI3K inhibitor monotherapy and, anticipating resistance to PI3K/mammalian target of rapamycin (mTOR) monotherapy, of combination therapies that include Parp inhibitors [9,10] or MEK inhibitors [11]. In a set of elegant experiments that tries to recapitulate clinical scenarios closely in vitro and in a mouse model, Britschgi and colleagues examined the biological basis for resistance to PI3K/mTOR inhibition in TNBC [12].
Britschgi and colleagues show that inhibition of PI3K not only rewires intracellular signaling but also leads cancer cells to recruit alternate extracellular signaling mechanisms to circumvent PI3K ( Figure 1). Th is inhibition occurs in a two-step process: within hours of exposure to the dual PI3K/mTOR inhibitor NVP-BEZ 235, TNBC cells responded with upregulation of insulinreceptor signaling and with its downstream eff ector IRS1 directly activating Janus kinase 2 (JAK2) and its substrate, the transcription factor signal transducer and activator of transcription 5 (STAT5). Presumably through chang ing the transcriptional profi le of the cancer cells, STAT5 then causes a more sustained upregulation of the IL-8 signaling axis, including secretion of IL-8 and upregulation of its receptor CXCR1 that then takes over to maintain JAK2/STAT5 signaling ( Figure 1). Th e net eff ect is that cancer cells which typically rely on receptor tyrosine kinases/PI3K signaling now shift to G-protein coupled receptors, in this case IL-8/CXCR1, to activate JAK2/STAT5 and to keep their mitotic machinery going. Th e biological signifi cance of this stepwise transition

Abstract
The phosphoinositide 3-kinase (PI3K) pathway serves as a relay where signals that emanate from the cell membrane are received and converted into intracellular signals that promote proliferation and survival. Inhibitors of PI3K hold promise for the treatment of breast cancer because activation of this pathway is highly prevalent. However, as is increasingly observed with inhibitors of cell signaling, there appear to be mechanisms of primary and secondary resistance. Britschgi and colleagues report that compensatory activation of the IL-8 signaling axis is a mechanism of primary resistance to PI3K inhibitors in some triplenegative breast cancers. In a set of experiments that carefully emulate the clinical scenario in a mouse model, they show that simultaneous inhibition of Janus kinase 2 enhances the effi cacy of PI3K/mammalian target of rapamycin inhibition. Their paper lends further support to the concept that successful design of treatments with signal transduction inhibitors must anticipate potential escape routes -and include agents to simultaneously block them. from receptor tyrosine kinases/PI3K to G-protein coupled receptor/JAK2 mitogenic signaling is confi rmed by the fi ndings that concomitant blockade of PI3K/mTOR and IL-8 signaling could eff ectively decrease tumor growth and metastasis and improve disease-free and overall survival in mice.
Britschgi and colleagues' fi ndings illustrate the plasticity of the signaling mechanisms that drive cancer cells: PI3K/ mTOR inhibition is acutely compensated for by the recruitment of IRS1/JAK2/STAT5 phosphorylation and eventually by a change of the transcriptional program in a way that leads to independence from PI3K signaling. Th ey show a pattern of resistance development where cancer cells immediately adapt with a change in phosphory lation routes, followed by transcriptional reprogram ming. For the practical purposes of cancer treatment the question really is just how many escape routes there are for cancer cells to evade monotherapy with a targeted agent, and specifi cally PI3K inhibition. Notably, while disease-free and overall survival was increased, none of the tumors in Britschgi and colleagues' model were cured by the PI3K/JAK2 inhibitor combination. Th is failure to cure disease means that tumor cells found a third way around PI3K inhibition, and it is intriguing to speculate that this third wave of resistance may be due to the evolution of genetically resistant clones that, for example, harbor a myc amplifi cation [13].
Will eff orts to treat breast cancer with combinations of signal transduction inhibitors be akin to the boy who is trying to hold the fl ood with his fi ngers in the dyke? Th e observed order of events with immediate adaptation of signaling pathways over transcriptional reprogramming and eventually presumed outgrowth of genetically resistant clones suggests that there may be a fi nite number of escape pathways, and inhibitors to target them may already be available. In addition, this novel approach might target specifi c tumor cell populations that are not typically eradicated by chemotherapy. Th e fact that combined JAK2 plus PI3K/mTOR inhibition greatly decreased circulating tumor cells, and specifi cally metastasis formation, suggests that this approach could potentially enhance the effi cacy of cytotoxic chemotherapy.
A number of fi ndings in Britschgi and colleagues' work raise immediate clinically important questions. Only some TNBC cell lines responded to PI3K inhibition with an increase in JAK2/phosphorylated-STAT5 signalingis there a common genetic denominator that dictates this response? Could measurement of blood IL-8 levels identify patients whose tumors might be de novo resistant to PI3K inhibition? Would concomitant blockade of IL-8 receptors, impor tant for neutrophil function, increase susceptibility to bacterial infections?
Rashes and non-infectious pneumonitis are known class eff ects that occur in patients treated with mTOR inhibitors [14], and may also occur with PI3K inhibitors. Given the signifi cance of IL-8 levels as a biomarker for pneumonitis, it is interesting to speculate that compensatory IL-8 production might actually be the mechanism for this thus far unexplained toxicity, and, if so, concomitant blockade of IL-8 signaling and PI3K might actually reduce pulmonary toxicity. Lastly, there is no reason to assume that upregulation of IL-8 signaling upon PI3K inhibition is specifi c to breast cancer, and upregulation of the IL-8 signaling axis should be considered a potential resistance mechanism in all cancers where PI3K inhibition is currently being studied.