API5 induces cisplatin resistance through FGFR signaling in human cancer cells

Abstract

Most tumors frequently undergo initial treatment with a chemotherapeutic agent but ultimately develop resistance, which limits the success of chemotherapies. As cisplatin exerts a high therapeutic effect in a variety of cancer types, it is often used in diverse strategies, such as neoadjuvant, adjuvant and combination chemotherapies. However, cisplatin resistance has often manifested regardless of cancer type, and it represents an unmet clinical need. Since we found that API5 expression was positively correlated with chemotherapy resistance in several specimens from patients with cervical cancer, we decided to investigate whether API5 is involved in the development of resistance after chemotherapy and to explore whether targeting API5 or its downstream effectors can reverse chemo-resistance. For this purpose, cisplatin-resistant cells (CaSki P3 CR) were established using three rounds of in vivo selection with cisplatin in a xenografted mouse. In the CaSki P3 CR cells, we observed that API5 acted as a chemo-resistant factor by rendering cancer cells resistant to cisplatin-induced apoptosis. Mechanistic investigations revealed that API5 mediated chemo-resistance by activating FGFR1 signaling, which led to Bim degradation. Importantly, FGFR1 inhibition using either an siRNA or a specific inhibitor disrupted cisplatin resistance in various types of API5^high cancer cells in an in vitro cell culture system as well as in an in vivo xenograft model. Thus, our results demonstrated that API5 promotes chemo-resistance and that targeting either API5 or its downstream FGFR1 effectors can sensitize chemo-refractory cancers.

Introduction

In general, traditional therapies such as surgery, chemotherapy and radiotherapy are implemented to treat many cancer types. Among these, chemotherapy is frequently used in diverse strategies, including neoadjuvant, adjuvant and combination chemotherapies.¹ Many patients with different types of cancers (for example, cervical, head and neck, non-small cell lung, gastric and bladder cancer) have been preferentially treated with cisplatin chemotherapy.^{2, 3, 25} These findings suggest the possibility that the effect of chemo-radiation therapy can be compromised in cervical cancer patients with API5 overexpression, an observation that can have profound clinical implications.

Here our study provided further evidence to satisfy an unmet clinical need. We elucidated the specific molecular mechanism for the close correlation between API5 overexpression and acquired resistance to cisplatin treatment. At first, the tumor cell population with high API5 expression levels steadily increased during the process of establishing a therapeutic resistance model using cisplatin (Figure 1). This result indicated that API5-overexpressing tumor cells are selected by repeated cisplatin treatments and develop resistance to this drug. There is evidence regarding the intimate relationship between API5 and cisplatin resistance. In addition, the crucial API5/FGFR/ERK axis that confers cisplatin resistance provides important clues suggesting that targeting molecules within the API5 downstream pathway can help overcome cisplatin resistance.

API5 contains protein-protein interaction domains such as HEAT and ARM repeats that are well suited for interactions with multiple binding partners.²⁸ Thus, discovering an API5 inhibitor using structure-based drug design studies is possible. However, there are no small-molecule drugs that directly target API5 to date. Considering this, we took note of an inhibitor of API5-mediated FGFR activity, which is a receptor tyrosine kinase that is part of a major class of drug targets, as a potential inhibitor of API5 signaling.^{29, 30, 31, 32} In a recent report, the small-molecule allosteric inhibitor SSR128129E, which blocks FGFR signaling at nanomolar doses, was identified,³³ and its therapeutic potential has been verified in immuno-resistant cancer cells.³⁴ It was determined whether this drug can attenuate the API5-mediated resistant properties induced by cisplatin in various types of aggressive cancer cells with high levels of API5 expression. The results were as predicted—when SSR128129E was used in combination with cisplatin in cisplatin-resistant API5^high cells, we observed that the cisplatin-mediated resistant phenotype disappeared. The observed synergistic effect of the combination treatment with cisplatin and SSR128129E is shown in Figure 5. This phenomenon may ultimately be driven by a dramatic increase in caspase 3 and Bim levels. Several studies have shown that cisplatin sensitivity is mediated by either Bim or caspase 3 regulation.^{35, 36} Thus, our results can be interpreted as a convergence of necrotic features via accumulation of cisplatin-induced apoptotic signals in cells with increased sensitivity to cisplatin via inhibition of FGFR signaling by SSR128129E.

Clinically, a therapeutic strategy with cisplatin that is effective against multiple types of cancers is necessary to minimize cisplatin resistance. In our study, we also conducted in vivo experiments to verify the potential of clinical application of this novel therapeutic strategy. We confirmed that SSR128129E significantly increased the therapeutic efficacy in an established xenograft mouse model using cisplatin-resistant human cancer cells when SSR128129E was used in combination with cisplatin. Thus, we consider it appropriate that during cisplatin-based chemotherapy, FGFR-targeted strategies are useful to treat refractory cancers as well as cisplatin-resistant cancers with high levels of API5 expression.