Etk Interaction with PFKFB4 Modulates Chemoresistance Of

Published OnlineFirst December 5, 2017; DOI: 10.1158/1078-0432.CCR-17-1475

Biology of Human Tumors Clinical Cancer Research Etk Interaction with PFKFB4 Modulates Chemoresistance of Small-cell Lung Cancer by Regulating Autophagy Qiongyao Wang1, Fanrui Zeng1, Yanqin Sun2, Qianqian Qiu1, Jian Zhang3, Weimei Huang1, Jie Huang4, Xiaomin Huang3, and Linlang Guo1

Abstract

Purpose: Epithelial and endothelial tyrosine kinase (Etk), also Results: Downregulation of Etk suppressed autophagy in che- known as bone marrow X kinase (Bmx), was found to be critical in moresistant SCLC cells, and direct inhibition of autophagy sen- modulating the chemoresistance of small-cell lung cancer (SCLC) sitized cells to chemotherapy. PFKFB4 (6-phosphofructo-2- in our preliminary study. However, the molecular mechanisms of kinase/fructose-2,6-biphosphatase 4) was identiﬁed as a down- Etk in SCLC chemoresistance remain poorly understood. stream target of Etk and an Etk-interacting protein, which pro- Experimental Design: We determined correlation of Etk with moted chemoresistance in SCLC and was associated with poor autophagy in SCLC. And direct inhibition of autophagy was therapeutic response and prognosis. Furthermore, ibrutinib was performed to validate its effect on chemoresistance. Coimmuno- found to exhibit a synergistic anti-tumor effect with chemother- precipitation (co-IP) and GST-pull down experiments were con- apy in targeting Etk. ducted to verify the interaction of Etk and PFKFB4, after a micro- Conclusions: Our results demonstrated for the ﬁrst time that array analysis. In vitro and in vivo gain or loss-of-function analyses Etk interacts with PFKFB4 to promote SCLC chemoresistance and evaluation of PFKFB4 expression in SCLC specimens, were through regulation of autophagy. Aberrant Etk and PFKFB4 can done to validate its role in chemoresistance. Ibrutinib was admin- be predictive factors for the chemotherapy response as well as istrated in SCLC cells to verify its synergistic anti-tumor effect with potential therapeutic targets in SCLC. Clin Cancer Res; 24(4); 950–62. chemotherapy using preclinical models including a PDX model. 2017 AACR.

Introduction develop multidrug resistance to chemotherapy (4, 5). Therefore, it is critical to elucidate the molecular mechanisms of chemore- Lung cancer is the leading cause of cancer-related death world- sistance and to identify effective therapeutic targets for SCLC. wide, with millions of new cases diagnosed annually (1). Small- Epithelial and endothelial tyrosine kinase (Etk), also known as cell lung cancer (SCLC) is a highly aggressive malignancy that bone marrow tyrosine kinase (Bmx), is a member of the Btk accounts for approximately 15%–18% of all lung cancers (2). (Bruton tyrosine kinase) family of kinases. The Btk family is SCLC is characterized by a rapid doubling time, the early devel- characterized by four conserved structural motifs: a pleckstrin opment of metastases, and a subsequently poor prognosis (3). A homology (PH) domain, Src homology 3 (SH3) and SH2 platinum–etoposide combination regimen remains the first treat- domains, and a catalytic tyrosine kinase domain (6). Specifically, ment option for SCLC, and majority of patients exhibit sensitivity Etk is expressed in epithelial and endothelial cells (including to this initial regimen. However, most SCLC patients rapidly cancer cells) rather than just in lympho-hematopoietic cells where Btk is expressed (7). Several studies have suggested that Etk plays significant modulatory roles in tumorigenicity, apoptosis and 1Department of Pathology, Zhujiang Hospital, Southern Medical University, treatment resistance of many human malignancies, including Guangzhou, P.R. China. 2Department of Pathology, Guangdong Medical Uni- prostate cancer, breast cancer, renal cell carcinoma, etc. (8–16). versity, Dongguan, P.R. China. 3Department of Oncology, Zhujiang Hospital, For example, Etk interacts with p53 in the cytoplasm to protect 4 Southern Medical University, Guangzhou, P.R. China. Guangdong Lung Cancer tumor cells from apoptosis and confers resistance to doxorubicin Institute, Guangdong General Hospital and Guangdong Academy of Medical in prostate cancer (10). In breast cancer, Etk promotes tumori- Sciences, Guangzhou, P.R. China. genic growth and resistance to antiestrogen therapies by phos- Note: Supplementary data for this article are available at Clinical Cancer phorylating Pak1 (P21-activated kinase 1; refs. 9, 16). Our pre- Research Online (http://clincancerres.aacrjournals.org/). vious study identified that Etk inhibits apoptosis and functions as Q. Wang and F. Zeng contributed equally to this article. a chemoresistance-associated protein by mediating either Bcl-2 or Corresponding Author: Linlang Guo, Department of Pathology, Zhujiang Hos- Bcl-XL in SCLC (17, 18). Another study showed that CTN06, a pital, Southern Medical University, 253 Gongye Road, Guangzhou 510282, P.R. dual inhibitor of Btk and Etk, induced autophagy and exhibited China. Phone: 8620-6278-3358; Fax: 8620-8431-1872; E-mail: increased sensitivity to docetaxel in prostate cancer cells, which [email protected] implied Etk might affect chemoresistance by modulating autop- doi: 10.1158/1078-0432.CCR-17-1475 hagy (19). Autophagy is a natural physiologic process for cellular 2017 American Association for Cancer Research. homeostasis to disassemble unnecessary or dysfunctional cellular

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Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

Translational Relevance Materials and Methods Clinical specimens The development of resistance to multiple chemotherapy A total of 68 formalin-fixed, paraffin-embedded (FFPE) tis- drugs has been a major clinical challenge in SCLC. Identifi- sues and 43 blood samples were collected from patients who cation of therapeutically actionable targets or pathways will had received bronchofiberscopy or biopsy for SCLC between pave the way for overcoming the challenge. In this study, we the January 2013 and October 2015 and received care and investigated the functional mechanism of Etk, a non-receptor follow-up in Zhujiang Hospital (Southern Medical University, tyrosine kinase, in SCLC chemoresistance and identified Guangzhou, China) or the First Affiliated Hospital of Guangz- PFKFB4 as a major downstream mediator. Our further studies hou Medical University (Guangzhou, China). Informed con- showed both Etk and PFKFB4 promote chemoresistance sent was obtained from all patients before specimen collection. through regulating autophagy. We also, for the first time, The experiments were approved by the Ethics Committee of the demonstrated that elevated levels of PFKFB4 were associated Southern Medical University (Guangzhou, China). All samples with poor chemotherapy response, suggesting that its expres- were independently reviewed by at least two pathologists and sion could serve as a valuable predictive factor for chemore- were divided as limited disease or extensive disease according sistance of SCLC. Our results from preclinical tumor models to the Veterans Administration Lung Study Group. Samples including patient-derived xenografts also demonstrated that were further distinguished as "sensitive" (complete response or ibrutinib, an Etk-related Btk inhibitor currently used in treat- partial response) and "refractory" (stable disease or progressive ing leukemia patients, strongly suppressed Etk activation in disease) groups according to Response Evaluation Criteria in SCLC cells and exerted a synergistic tumor-inhibitory effect Solid Tumors (RECIST Edition 1.1). with chemotherapeutic drugs, thus nominating ibrutinib as a candidate therapeutic agent for chemoresistant SCLC patients. Cell lines Human SCLC cell lines NCI-H69, NCI-H446, and the chemoresistant H69AR were purchased from ATCC. Human SCLC components, which plays a dual role in cancer (20), and accu- cell lines NCI-H82, H209, H345, H146, and H526 were obtained mulating researches suggested that autophagy facilitates resis- as a generous gift from Dr. Ji Lin of MD Anderson Cancer Center tance to chemotherapy in many human tumors (21–24). Etk was (Houston, TX). The other drug-resistant subline, H446DDP, was identified as an important chemoresistance-associated protein in established in our laboratory by culturing H446 cells in cisplatin SCLC (17, 18), but the molecular mechanisms of Etk leading to and is described in detail in the Supplementary Materials and the poor therapeutic response remains obscure. Methods section. 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) belongs to a family of bifunctional PFKFB enzymes, Plasmids and cell transfection which regulates glycolysis by controlling fructose-2,6-bispho- Cells were transiently transfected with validated siRNAs for Etk sphate (F2,6-BP) levels (25, 26). Overexpression of PFKFB4 was and PFKFB4 and corresponding negative control (GenePharma) observed in many human cancers (27–29), with the majority of and the lentiviral particles of shEtk, Etk-GFP, or shPFKFB4, studies focusing on glycolytic flux modulation by PFKFB4 during PFKFB4-GFP (GenePharma). Detailed descriptions of the exper- hypoxia (30–35). Strohecker and colleagues recently identified iment can be found in the Supplementary Materials and Methods PFKFB4 as a novel autophagy regulator by a high-throughput section. shRNA screen after deleting the autophagy cargo receptor and substrate p62/SQSTM1 (36). Knockdown of PFKFB4 increased RNA isolation, real-time qRT-PCR, Western blot, flow autophagic flux (36). This report revealed the function of PFKFB4 cytometry, and cell counting kit-8 (CCK-8) assays on autophagy, which implied us that PFKFB4 might modulate RNA isolation, real-time qRT-PCR, Western blot analysis, flow autophagy in SCLC. cytometry, and Cell counting kit-8 (CCK-8) assay were performed As the reports that implied a relationship between Etk, as described previously (37), and as detailed in the Supplemen- chemoresistance, and autophagy, we hypothesized that Etk tary Materials and Methods section. may contribute to chemoresistance in SCLC by modulating autophagy, which is validated in the following tests, and Ibrutinib inhibition of autophagy sensitized the SCLC cells to chemo- Ibrutinib (PCI-32765) was purchased from Selleck Biochem- therapy. Furthermore, we performed a microarray analysis to icals as a powder. For Western blot analysis, ibrutinib was dis- identify downstream effectors of Etk that may affect autophagy. solved in DMSO and diluted to 1, 2, or 5 mmol/L in culture media. After screening, we identified PFKFB4 expression was associated For animal experiments, ibrutinib powder was dissolved in a 10% with Etk and its contribution to autophagy in SCLC. Besides, we HP-beta-cyclodextrin solution (1.6 mg/mL) and administered to validated the direct interaction of PFKFB4 and Etk in protein mice in the drinking water. level. Then, we investigated the biological function of PFKFB4 on the chemotherapy response both in vivo and in vitro,andits IHC expression associated with various clinicopathologic character- Tissues of SCLC clinical samples were fixed in 4% paraformal- istics. Taken together, our results identify a novel mechanism of dehyde, and then embedded in paraffin blocks. Four-micron- Etk in promoting chemoresistance through regulating autop- thick sections were cut and analyzed for PFKFB4 (Abcam) or Etk hagy, suggesting the potential application of PFKFB4 expres- (Abcam) protein expression. The slides were incubated in PFKFB4 sion in predicting drug resistance and in customizing optimal antibody or Etk antibody diluted to 1:100 at 4C overnight and treatments of SCLC. then with the secondary antibodies for 2 hours. The sections were

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viewed under EnVision peroxidase system (Dako) and then uranyl acetate and lead citrate. Digital images obtained using analyzed with Image-Pro Plus analysis software. Philips CM-120 Transmission Electron Microscopy at 60 kV.

cDNA expression microarray Tumor xenograft experiments cDNA expression microarray was performed as described pre- Tumor xenograft experiments were performed as described viously (38), and as detailed in the Supplementary Materials and previously (37), and as detailed in the Supplementary Materials Methods section. and Methods section.

Coimmunoprecipitation Generation of patient-derived xenografts m Total of 1 mg whole cell was incubated overnight with 2 g Fresh tissues from a chemo-na€ve SCLC patient undergoing m of antibodies against Etk (Abcam). Then, 100 Lpreblocked surgical treatment were obtained and transported immediately to agarose beads was added to the antibody/lysate mixture and the animal facility in PBS at 4 C. Primary tissue sample was incubated for another 2 hours at 4 C. After incubation, immu- anonymized and obtained in accordance with the Zhujiang nocomplexes were washed for three times in HEGNDT buffer Hospital of Southern Medical University (Guangzhou, China) [10 mmol/L HEPES (pH 8.0), 1 mmol/L EDTA, 10% glycerol, Institutional Review Board. Primary SCLC tissue specimens were 50 mmol/L NaCl, 2 mmol/L dithiothreitol (DTT)] containing minced with scissors into small (2–3mm3) fragments. Under 0.1% Triton X-100 and once in HEGNDT buffer without Triton aseptic conditions, tissue fragments were implanted subcutane- fi X-100.Theproteincomplexwas nally eluted off the beads for ously into the flanks of female B-NSG mouse (NOD-Prkdcscid Western blot analysis, and the solubilized proteins were sub- Il2rgtm1/Bcgen, BIOCYTOGEN) by using a 10-gauge Trochar fl jected to SDS-PAGE, transferred onto a polyvinylidene di uor- needle through a small incision on the animal's dorsal flank. ide membrane, and analyzed by Western blotting using the Animal health was monitored daily. Once established, solid antibody of PFKFB4. tumor xenografts were serially passaged using the same technique.

GST-pull-down assay Statistical analysis Etk cDNA was isolated by RT-PCR and cloned into the XhoI and Data are represented as mean SD of at least three independent EcoRI sites of GST-tagged pGEX-4T-1 vector by T4 DNA Ligase experiments. Independent samples t-test or one-way ANOVA was (Thermo Scientific), whereas PFKFB4 cDNA was cloned into the employed to analyze the possible differences between groups. The XhoI and EcoRI sites of His-tagged pET-28a(þ)vector. The pGEX- association between PFKFB4 and Etk expression was analyzed by 4T-1-Etk and pET-28a(þ)-PFKFB4 recombinant plasmids were Spearman rank correlation test. The association between PFKFB4, transformed into E. coli BL21(DE3) separately. PCR identification, Etk expression, and clinicopathologic characteristics was explored double enzyme digesting, and sequencing were used to screen and by x2 test. Survival curves were estimated using the Kaplan–Meier identify the high expressing positive clone, which synthesized analysis. Prognostic factors were evaluated by univariate and GST-Etk or His-PFKFB4 recombinant proteins. The purified GST- multivariate analyses (Cox proportional hazards model). Differ- Etk fusion proteins were attached to Glutathione Sepharose (GE ences with P values of less than 0.05 were considered statistically Healthcare), and then mixed and incubated with the purified His- significant. All statistical analyses were performed using SPSS 17.0 PFKFB 4 protein at 4C overnight. The eluted samples were software. detected by Western blot analysis using the GST antibody (Trans- Gen) and His antibody (CWBio). Results mRFP-GFP-LC3 analysis and confocal laser scanning Chemoresistant SCLC cells exhibit increased autophagy microscopy To test our hypothesis that Etk regulates the chemoresistance The adenoviral particles of tandem mRFP-GFP-LC3 were pur- of SCLC by modulating autophagy, we first examined whether chased from Hanbio. SCLC cells (approximately 1 CL 103) were chemoresistant SCLC cells exhibited increased autophagy. seeded into glass bottom cell culture dish (NEST). Twenty-four Autophagosomes in SCLC were evaluated using transmission hours after mRFP-GFP-LC3 adenovirus infection, cells were trans- electron microscopy (TEM) and confocal laser scanning micros- fected with siRNA targeting PFKFB4, Etk, or the corresponding copy (CLSM), as well as Western blotting to detect the conver- negative control (siNC). The next day, cells were fixed with sion of LC3-I to active LC3-II protein (i.e., the LC3-II/LC3-I formaldehyde for 20 minutes. After being rinsed in PBS, the ratio) and P62 protein levels, two hallmarks of autophagy. As nuclei were stained with DAPI (Beyotime) for 5 minutes. Spe- shown in Fig. 1A, TEM detected an increased number of cially, suspension cell line H69 was analyzed directly without autophagosomes in H69AR, H446DDP, and H82 cells than treated with formaldehyde and DAPI. The cells were then ana- that in H69, H446, and H209. As shown in Fig. 1B, Western lyzed using LSM880 laser scanning confocal microscope (Zeiss). blotting demonstrated that compared with the drug-sensitive The number of green or red punctate fluorescence in each cell was cells, H69AR, H446DDP, and H82 cells had an increased LC3- counted in 10 different fields under the confocal laser scanning II/LC3-I ratio and a decreased P62 protein level, suggesting that microscope. the chemoresistant SCLC cells exhibited increased autophagy. Moreover, we expressed in the SCLC cells an mRFP-GFP-LC3 Transmission electron microscopy fusion protein using adenovirus vectors. Cells were fixed with 2.5% glutaraldehyde containing CLSM analysis showed an increased mRFP-GFP-LC3 signal in 0.1 mol/L sodium cacodylate, and treated with 1% osmium H69AR, H446DDP, and H82 compared with their parental sen- tetroxide. After dehydration, samples were embedded in ara- sitive cells, indicating an increase in autophagy concurrent with ldite and then cut into thin sections that were stained with the development of chemoresistance (Fig. 1C). Together, the

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Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

Figure 1. Knockdown of Etk suppresses autophagy and inhibition of autophagy restores the chemosensitivity of SCLC cells. A, TEM evaluated autophagy in SCLC cells. B, Western blot analysis of LC3II/I and p62 expression. C, CLSM analysis of LC3 puncta in SCLC cells that transiently express the mRFP-GFP-LC3 fusion protein. D and E, Western blot analysis and CLSM were used to examine the effect of siRNA against Etk on autophagy in H69AR and H446DDP cells. F, Western blot analysis of LC3II/I expression after chloroquine (CQ) administration (20 mmol/L) for 24 hours in H69AR and H446DDP cells. G, CCK-8 assays showed chloroquine or si-RNA targeting Etk signiﬁcantly decreased IC50 values to chemotherapeutic drugs (i.e., adriamycin, ADM; cisplatin, CDDP; etoposide, VP-16) of H69AR (left) and H446DDP (right). Error bars, mean SD from three independent experiments. , P < 0.05; , P < 0.01. Scale bars, 5 mm.

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Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

above results suggest that chemoresistant SCLC cells exhibit in H69AR, H446DDP, and H82 cells, whereas restoration of Etk increased autophagy. led to the opposite changes in the chemosensitive cells (Fig. 2B and C; Supplementary Fig. S2A). The above results indicate that Etk mediates SCLC chemoresistance through controlling Etk signaling stimulates the expression of PFKFB4. autophagy To further explore the function of Etk in control of PFKFB4, we Etk was identified as an important chemoresistance-associated examined whether the two proteins associate with each other. protein in SCLC in our previous study, which showed that Indeed, our coimmunoprecipitation (co-IP) assay detected a knockdown of Etk impaired resistance of H69AR cells to chemo- significant amount of PFKFB4 protein being associated with Etk therapeutic drugs (18). Study by Guo and colleagues showed that (Fig. 2D). CTN06, a dual inhibitor of Btk and Etk, induced autophagy in To evaluate whether these two proteins directly interacted with prostate cancer cells (19), implicating a role of Etk in modulating each other, we performed glutathione-S-transferase (GST)-pull autophagy. Besides two pairs of chemoresistant SCLC cell lines, down assay with purified GST-tagged Etk and His-tagged PFKFB4 the other five SCLC cell lines including H82, H209, H526, H146, Results from the pull-down assay showed that purified His- and H345 were tested by CCK-8 assays to screen their relative PFKFB4 protein was associated with GST-Etk, but not GST (Fig. chemoresistance. H82 was regarded as the comparative chemore- 2E). Together, the results indicate that Etk directly interacts with sistant cells with highest IC50 values and H209 as chemosensitive PFKFB4 and that their expression is correlated in SCLC. cells with lowest IC50 values (Supplementary Fig. S1A). To determine that Etk plays a function in control of autophagy, PFKFB4 controls autophagy in SCLC cells we knocked down its expression in SCLC cells using siRNA and A recent study identified PFKFB4 as a novel autophagy regulator then analyzed the effects on autophagic flux. As shown in Sup- by a high-throughput shRNA screening with murine immortal- plementary Fig. S1C, Etk mRNA and protein expression is signif- ized epithelial cells (36). To examine whether PFKFB4 regulates icantly elevated in H69AR, H446DDP, H82 cells compared with autophagy in SCLC cells, we knocked down its expression in SCLC H69, H446, and H209 cells. The efficiency of siRNA knockdown cells and then analyzed the autophagic flux. The efficiency of in H69AR and H446DDP cells was confirmed using qRT-PCR and siRNA knockdown in H69AR, H446DDP, and H82 cells was Western blotting (Supplementary Fig. S1D and S1E). Western confirmed using qRT-PCR (Supplementary Fig. S3A and S3B). blotting analysis and CLSM revealed that knockdown of Etk TEM revealed that the number of autophagic vesicles was inhibited autophagy in drug-resistant SCLC cells (Fig. 1D and E). decreased in H69AR and H446DDP cells transfected with siRNAs To further examine the contribution of autophagy to the targeting PFKFB4 (Supplementary Fig. S3C). Similarly, Western chemoresistance of SCLC, chloroquine, which inhibits autopha- blotting and CLSM analysis revealed that knockdown of PFKFB4 gic cargo degradation, was used to block autophagy. Chloroquine inhibited autophagy in drug-resistant SCLC cells (Fig. 2F and G), was added to cells for 24 hours at a final concentration of 20 indicating that indeed in SCLC cells, PFKFB4 regulates autophagy. mmol/L. Western blotting revealed an increased LC3II/LC3I ratio To determine whether Etk controls autophagy through PFKFB4, in H69AR and H446DDP cells after chloroquine treatment (Fig. we performed rescue experiments. Results obtained from the 1F). Moreover, the CCK-8 assays revealed that chloroquine sig- experiments demonstrated Etk overexpression increased autop- nificantly inhibited drug resistance of H69AR and H446DDP cells hagy; however, the increased authophagy was diminished by (Fig. 1G). Taken together, these results demonstrate that chloro- knockdown of PFKFB4 (Fig. 2H and I). quine and siRNAs targeting Etk could inhibit autophagy and enhance the sensitivity of resistant SCLC cells to chemotherapeu- PFKFB4 promotes chemoresistance of SCLC in vitro and in vivo tic agents. Next, to investigate whether PFKFB4 plays a role in chemoresistance in SCLC, we first analyzed PFKFB4 expression in che- Etk directly interacts with PFKFB4 moresistant and chemosensitive SCLC cell lines. Intriguingly, To explore the molecular targets associated with Etk, a cDNA chemoresistant H69AR, H446DDP, and H82 cells expressed microarray analysis was conducted using H69AR cells transfected significantly higher levels of PFKFB4 than the chemosensitive with siRNA targeting Etk. A total of 25 genes were downregulated, H69, H446, and H209 cells, at both mRNA and protein and 31 genes were upregulated by more than 1.5-fold in si-Etk– level (Supplementary Fig. S4A). In order to assess whether treated cells (Fig. 2A). Among those genes, PFKFB4 caught our PFKFB4 was functionally involved in the chemoresistance of attention because of its association with the tumor growth in lung SCLC, we altered PFKFB4 expression in the cells by shRNA cancer, prostate cancer, breast cancer, etc. (34, 35). (shPFKFB4-1, shPFKFB4-2)–mediated stable knockdown in Consistent with the microarray data, knockdown of Etk indeed H69AR, H446DDP, and H82 cell lines or PFKFB4 overexpression inhibited PFKFB4 expression at both the mRNA and protein level (LV5-PFKFB4) in chemosensitive SCLC cells (Supplementary

Figure 2. PFKFB4 interacts with Etk and affects autophagy of SCLC. A, Differentially expressed genes in H69AR-siNC cells versus H69AR-siEtk-1 cells were evaluated by cDNA microarray (1.5-fold). B, qRT-PCR and Western blot analysis of PFKFB4 expression in H69 cells transfected with pcDNA3.1-Etk or negative control vector (one-way ANOVA). C, qRT-PCR and Western blot analysis of PFKFB4 expression in H69AR cells transfected with siRNA targeting Etk. D, The interaction between Etk and PFKFB4 was analyzed by co-IP in H69AR cells. Anti-Etk antibody was used for IP. The amounts of PFKFB4 in the immunoprecipitates were detected by Western blot analysis. E, Etk binds to PFKFB4 in vitro directly. Puriﬁed bacterially expressed His-PFKFB4 fusion proteins were mixed with puriﬁed bacterially expressed GST or GST-Etk fusion proteins. The immunoprecipitated His-tagged fusion proteins were detected by Western blot analysis with anti-His antibody. The bottom panels showed the inputs of His-PFKFB4 and GST-Etk fusion proteins in immunoprecipitation experiment. F and G, Western blot and CLSM were used to examine the effect of siRNA against PFKFB4 on autophagy in H69AR, H446DDP, and H82 cells. H and I, LC3II/I expression of H69 cells were detected by Western blot analysis and CLSM, after transfected with pcDNA3.1-Etk, siRNA targeting PFKFB4, or their corresponding negative control vector. Error bars, mean SD from three independent experiments. , P < 0.05; , P < 0.01.

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Figure 3.

PFKFB4 represses sensitivities to anticancer drugs of SCLC cells in vitro and in vivo. A and B, CCK-8 assays showed PFKFB4 overexpression increased IC50 values to chemotherapeutic agents of H69, H446, and H209 cells, whereas knockdown of PFKFB4 decreased IC50 values of H69AR, H446DDP, and H82 cells (one-way ANOVA). C, IC50 values of H69 cells were tested by CCK-8 assays, after being transfected with pcDNA3.1-Etk, siRNA targeting PFKFB4, or their corresponding negative control vector. D, Effect of PFKFB4 on sensitivities to chemotherapeutic agents in nude mice. H446 cells stably overexpressing PFKFB4 or the control, whereas H446DDP cells were stably transfected with shControl or shPFKFB4-1. Each group of cells were injected into mice, afterwards drugs (CDDP þ VP-16) or vehicles were injected intraperitoneally as indicated (n ¼ 5 mice for each group). E, The growth curve of tumor volumes of the PFKFB4 overexpression groups or PFKFB4 knockdown groups. F and G, Flow cytometry was performed to evaluate the impact of PFKFB4 on apoptosis induced by ADM. SCLC cells were stained with V450 and Annexin V after treatment with antineoplastic drugs. Early and late apoptotic cells are shown in the right quadrant. Error bars, mean SD from three independent experiments. , P < 0.05; , P < 0.01.

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Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

Figure 4. PFKFB4 was inversely correlated with poor chemotherapy response and prognosis in SCLC patients. A and C, Representative IHC staining of Etk or PFKFB4 in normal alveolar epithelium, chemosensitive, and refractory SCLC FFPE tissues (magnification 400). B and D, Etk- or PFKFB4-positive expression rate was frequently increased in chemoresistant SCLC tissues compared with the chemosensitive SCLC tissues and normal alveolar epithelium. E, PFKFB4 and Etk protein expression was tested by IHC in 68 SCLC samples. The correlation coefficient between expression levels of PFKFB4 and Etk was analyzed using Spearman correlation (r: correlation coefficient; P ¼ 0.0002). F, Kaplan–Meier analysis of overall survival of 68 patients with SCLC based on PFKFB4 expression. , negative; þ, positive. G, The mRNA PFKFB4 expression in blood sample of drug-refractory SCLC (n ¼ 19) and -chemosensitive SCLC patients (n ¼ 24) was detected using qRT-PCR. Scale bars, 20 mm., P < 0.05; , P < 0.01.

Fig. S4B and S4C). Furthermore, we were able to rescue the shRNA with Etk overexpression exhibited increased IC50 values for the knockdown of PFKFB4 by ectopic expression of PFKFB4 cDNA chemotherapeutic drugs tested (Fig. 3C), and knockdown of that was insensitive to shPFKFB4 (Supplementary Fig. S4F). We PFKFB4 in H69 cells could rescue the increase of the IC50 values then examined the effects of PFKFB4 expression alteration on the mediated by Etk upregulation (Fig. 3C; Supplementary Fig. S2D chemoresistance in SCLC cells. CCK-8 assays were performed to and S2E). measure the sensitivity of SCLC cells to various chemotherapeutic Moreover, to determine whether PFKFB4 confers chemoresis- drugs (i.e., adriamycin, ADM; cisplatin, CDDP; etoposide, VP- tance in vivo, we subcutaneously transplanted H446 and 16). The results revealed that the IC50 values (i.e., the concentra- H446DDP cells with altered PFKFB4 expression into nude mice. tion at which growth is inhibited by 50%) were increased in PFKFB4 expression in the tumor xenografts was measured by IHC chemosensitive SCLC cells with PFKFB4 overexpression (Fig. 3A), (Supplementary Fig. S4D and S4E). PFKFB4 knockdown signif- whereas the IC50 values of H69AR, H446DDP, and H82 cells were icantly reduced the tumor volumes after chemotherapy treatment signiﬁcantly decreased after knockdown of PFKFB4 (Fig. 3B; (CDDP and VP-16); in contrast, the tumor volumes were obvi- Supplementary Fig. S4G). ously larger in the PFKFB4 overexpression group than those in the To further conﬁrm that PFKFB4 is a major downstream corresponding control groups (Fig. 3D and E). These results effector of Etk in SCLC chemoresistance, CCK-8 assays were suggested that PFKFB4 could affect the chemosensitivity of SCLC used to evaluate the effects of knockdown of PFKFB4 in Etk- cells in vivo. overexpressing cells on chemo-drug sensitivity. The results We next evaluated the effect of PFKFB4 on apoptotic cell death showed that, compared with the empty vector controls, cells upon cell exposure to chemotherapeutic drugs. Flow cytometry

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Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

analysis of Annexin V demonstrated that PFKFB4 knockdown in overexpression was correlated with poor survival, worse response H69AR and H446DDP cells led to increased apoptosis after to chemotherapy, and later stages in SCLC. treatment with ADM, CDDP, or VP-16, whereas PFKFB4 overexpression in H69 and H446 cells decreased apoptosis (Fig. 3F Ibrutinib suppresses chemoresistance of SCLC and G; Supplementary Fig. S4H and S4I). Ibrutinib (PCI-32765), an irreversible phosphorylation inhibitor of Btk, inactivates the B-cell antigen receptor (BCR) Elevated PFKFB4 expression correlates with poor survival and signaling pathway, which is vital for the survival of malignant B chemotherapy response in SCLC patients cells (39). Oral administration of ibrutinib is highly effective in To determine the clinicopathologic significance of PFKFB4 controlling mantle cell lymphoma and certain types of chronic expression in SCLC and clinical correlation between Etk and lymphocytic leukemia (40). As a member of a non-receptor PFKFB4, 68 cancer specimens and 10 normal lung tissues were tyrosine kinase family, Etk shares a similar catalytic tyrosine analyzed using IHC. The intensity of PFKFB4 staining in the kinase domain with Btk and is partially inhibited by ibrutinib. chemoresistant SCLC specimens was much stronger than that In light of the high clinical efficacy of ibrutinib in treating B-cell in the drug-sensitive group, and weak expression was detected malignancy, we hypothesized that ibrutinib may affect the in normal lung alveolar cells (Fig. 4A). In addition, positive chemosensitivity in SCLC cells by suppressing Etk activation. PFKFB4 expression was detected in 26 of the 32 (81.25%) Because ibrutinib inhibits phosphorylation of the target, we chemoresistant SCLC samples, compared with 20 of the 36 first evaluated expression levels of Etk and tyrosine-phosphor- (55.56%) drug-sensitive SCLC tissues and 2 of the 10 normal ylated Etk (pEtk) in SCLC cells (Supplementary Fig. S1B; Fig. alveolar epithelial cells (20%; Fig. 4B). Similarly, elevated Etk 5A). Using H69AR (which express higher levels of Etk and pEtk) expression was detected in the drug-sensitive group compared and H69 cells as models, we added ibrutinib at doses ranging with the drug-resistant group (Fig. 4C). The rate of Etk posi- from 0.1 mmol/L to 5 mmol/L for 24 hours. The Western tivitywasmuchhigherinanticancerdrug–refractory SCLC blotting analysis revealed that ibrutinib modestly inhibited specimens (87.5%) than in drug-sensitive specimens Etk expression and that 1 mmol/L ibrutinib significantly (52.78%) and normal bronchial epithelium (10%; Fig. 4D). decreased pEtk expression in H69AR cells (Fig. 5B left), whereas As shown in Supplementary Table S1, compared with the drug- no significant changes were observed in H69 cells (Fig. 5B sensitive group, the chemoresistant group had a significantly right). The CCK-8 assays revealed that the IC50 values of the higher frequency of positive PFKFB4 expression (P ¼ 0.006), chemotherapeutic drugs were significantly decreased in H69AR which was also higher in the extensive stages group, compared cells treated with 1 mmol/L ibrutinib (Fig. 5C left) However, no with the earlier stages one (P ¼ 0.01). significant difference of the IC50 values was observed in H69 However, there were no significant differences with respect to cells treated with ibrutinib (Fig. 5C right). gender or age (56 years and >56 years; Supplementary Table S1). In addition, to determine whether ibrutinib modulates che- In terms of Etk expression in SCLC specimens, the higher Etk moresistance in vivo, we established and used a patient-derived expression was correlated with a poor chemotherapy response xenograft (PDX) model with tumor tissues from a SCLC patient and extensive disease stage (Supplementary Table S1). Further- and also a subcutaneous xenograft models of H69 and H69AR. more, we compared Etk and PFKFB4 expression using IHC in The tumor-carrying mice were administered either ibrutinib (25 SCLC tissues prior to initial treatment and found that PFKFB4 mg/kg daily) in their drinking water, or chemotherapeutic levels were positively correlated to Etk expression (Fig. 4E), which drugs (CDDPþVP-16) by intraperitoneal injection, or the com- was in agreement with their expression in peripheral blood using bination. The clinicopathologic characteristics of the patient qRT-PCR (Supplementary Fig. S2B and S2C). Kaplan–Meier sur- that donated SCLC specimen to perform PDX experiment is vival analysis demonstrated high levels of PFKFB4 expression listed in Supplementary Table S3. The results showed a smaller were correlated with a shorter overall survival in SCLC patients tumor volume in the ibrutinib or chemotherapeutic-treated (Fig. 4F). The multivariate analysis revealed that PFKFB4 was an mice, but combination of the two treatments reduced the independent prognostic factor (P ¼ 0.004; Supplementary Table tumor volume significantly in the H69AR tumor model (Fig. S2). In addition, we tested circulating PFKFB4 levels in peripheral 5D and E, left). Interestingly, whereas no significant changes blood, which was drawn from SCLC patients prior to initiating were observed in the H69 tumors (Fig. 5D and E, right), IHC therapy. The result indicated that PFKFB4 expression was higher in analysis revealed significantly reduced expression of Etk and the drug-resistant group (n ¼ 19) than in the drug-sensitive group pEtk in the xenografts of ibrutinib-treated group (Fig. 5F). (n ¼ 24; Fig. 4G). Collectively, these results indicate that PFKFB4 Significantly, the PDX tumors displayed similar results. As

Figure 5. Ibrutinib restores chemosensitivity of SCLC cells. A, Expression of phosphorylated Etk (pEtk) was evaluated by Western blot analysis in SCLC cell lines H69, H69AR, H446, and H446DDP. B, Western blot analysis showed ibrutinib modestly reduced expression of Etk (left top) and 1 mmol/L ibrutinib significantly decreased pEtk expression in H69AR (left bottom), whereas no significant difference in H69 (right). H69AR and H69 cells were treated with DMSO or ibrutinib (dose gradient: 0.1 mmol/L, 1 mmol/L, 5 mmol/L) for 24 hours. C, CCK-8 assays showed the effect of ibrutinib (1 mmol/L) on IC50 values to anticancer drugs of H69AR (left) and H69 (right) cells. D, Effect of ibrutinib, chemotherapy (CDDPþVP-16), or combination of ibrutinib and chemotherapy on tumor growth of SCLC cells in vivo. The nude mice were engrafted with H69AR or H69 cells subcutaneously. At day 7, mice were randomized according to tumor volume to receive vehicle (HP-beta-cyclodextrin), ibrutinib (25 mg/kg/day), chemotherapy (CDDP 3 mg/kg and VP-16 7 mg/kg). E, Growth curve of tumor volumes in each group of H69AR (left) or H69 (right; n ¼ 4). F, Representative IHC staining of Etk and pEtk in tumor xenografts of ibrutinib group or vehicle group. G, Effect of ibrutinib, chemotherapy (CDDP þ VP-16), or combination of ibrutinib and chemotherapy on tumor growth using SCLC PDX models (n ¼ 3). H, Growth curve of tumor volumes in each group of PDX models (n ¼ 3). I, Histopathologic features and representative IHC staining of Syn, CD56 in a SCLC patient tumor and corresponding PDX xenografts (magnification 400). J, Expression of Etk and pEtk in PDX xenografts of ibrutinib group or vehicle group were evaluated by IHC staining. , P < 0.05; , P < 0.01 compared with control.

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Wang et al.

showninFig.5GandH,combinedtreatmentofibrutiniband tinib (Fig. 6C). Phosphorylation of Etk at Y566 has been charac- chemotherapy signiﬁcantly decreased the tumor volume. terized as a hallmark of Etk activation. Therefore, the selective Expression of Etk and pEtk was inhibited with ibrutinib admin- inhibition by ibrutinib of Etk phosphorylation at Y566, but not istration (Fig. 5J). IHC analysis showed similar expression of Btk, in SCLC cells strongly suggests that ibrutinib targets Etk Syn and CD56 and histomorphology between the SCLC patient function in SCLC cells. specimen and the corresponding PDX xenograft tumors (Fig. To further demonstrate that the cell growth inhibition effect by 5I). Collectively, these results suggested a potentially synergistic ibrutinib is mediated by targeting Etk, we next performed CCK-8 effect of ibrutinib with chemotherapy in SCLC tumors. assays. As shown in Fig. 6D, ibrutinib strongly sensitized H69AR cells to the three different chemotherapy drugs (i.e., ADM, CDDP, and VP-16) indicated by the drug IC decreases in cells treated Ibrutinib suppresses chemoresistance by inhibition of Etk 50 with ibrutinib (1 mmol/L). Remarkably, Etk ectopic expression activation could effectively diminish the IC reduction to the three chemo- To explore the mechanism of action of ibrutinib, we performed 50 drugs. Taking together, the results suggest that ibrutinib sup- rescue experiments to determine whether ibrutinib works by presses chemoresistance by inhibiting Etk activation in SCLC. inhibiting Etk or also Btk. As shown in Fig. 6A and B, the expression of Btk, especially phosphor-Btk (pBtk), is very low in the SCLC cells examined. Importantly, ibrutinib treatment at Discussion doses ranging from 0.1 mmol/L to 5 mmol/L had no signiﬁcant Etk was found to be an important chemoresistance-associated effect on Btk and pBtk expression. In contrast, ibrutinib effectively protein by mediating Bcl-XL and inhibited apoptosis via Bcl-2 of inhibited Etk and Y566-phosphor-Etk (pEtk) expression in the SCLC in our previous study (17, 18). However, the molecular SCLC cells, whereas restoration of Etk rescues the effect of ibru- mechanisms underlying chemoresistance mediated by Etk in

Figure 6. Ibrutinib regulates chemoresistance by suppressing pEtk in SCLC. A, Expression of Btk and phosphorylated Btk (pBtk) was evaluated by qPCR and Western blot analysis in SCLC cell lines H69, H69AR, H446, and H446DDP. B, Western blot analysis was used to evaluate the expression of Btk and pBtk in H69AR cells after ibrutinib administration (dose gradient: 0.1 mmol/L, 1 mmol/L, 5 mmol/L) for 24 hours. C, The expression of Etk and pEtk were detected by Western blot analysis,

after transfected with pcDNA3.1-Etk, or the corresponding negative control vector with adding ibrutinib in H69AR cells. D, IC50 values of H69AR cells were tested by CCK-8 assays, after being transfected with pcDNA3.1-Etk or their corresponding negative control vector with ibrutinib administration (1 mmol/L) simultaneously. Error bars, mean SD from three independent experiments. , P < 0.05; , P < 0.01 compared with control. E, Schematic diagram shows Etk and PFKFB4 regulate chemoresistance in SCLC.

960 Clin Cancer Res; 24(4) February 15, 2018 Clinical Cancer Research

Etk Controls PFKFB4 in Promoting Chemoresistance of SCLC

SCLC remained elusive. In this study, we demonstrated for the first ibrutinib modestly inhibited Etk expression and that an appro- time that Etk interacts with PFKFB4 to promote the chemoresis- priate concentration of ibrutinib significantly decreased pEtk tance of SCLC by modulating autophagy (Fig. 6E). expression in vivo and in vitro. Ibrutinib administration did not To further understand the biological function of Etk in significantly affect tumor growth in a nude mouse model, which is chemoresistance of SCLC, we examined the literatures. A study consistent with the high IC50 values of ibrutinib observed in SCLC showed that CTN06, a dual inhibitor of Btk and Etk, promoted cells (data not shown). However, the combination therapy of autophagy in prostate cancer, which also exhibited increased ibrutinib and anticancer drugs significantly reduced the tumor sensitivity to docetaxel and implied a negative correlation volumes, which suggests a synergistic effect of ibrutinib to che- between Etk and autophagy (19). Thus, we hypothesized that motherapy in SCLC cells. This study implied the potential use of Etk modulated chemoresistance of SCLC by autophagy. To ibrutinib in SCLC. To further investigate molecular mechanisms confirm this, we compared the autophagic flux in SCLC cells about how ibrutinib enhances the effect of antineoplastic drugs in and found that chemoresistant cells exhibited increased autop- killing SCLC cells, additional tests are required. hagy and knockdown of Etk inhibited autophagy. Then we Lack of preclinical tools for predicting clinical activity of novel performed a microarray analysis to screen molecules down- therapeutic strategies in cancer restricts the development of prog- stream of Etk and figured out that PFKFB4, as a downstream ress in oncology. More and more evidence shows that PDX has signal, might modulate chemoresistance of SCLC by regulating been a promising powerful tool to overcome this shortcoming (5, autophagy. 41–44). To further support that ibrutinib restores chemosensi- Majority of studies about PFKFB4 focus on glycolytic flux tivity of SCLC, we used a PDX model and showed the similar modulation (30–35). Recently, Strohecker and colleagues iden- results as SCLC cell line xenografts experiments, which suggest the tified PFKFB4 as a novel autophagy regulator by a high-through- potential clinical application of ibrutinib in SCLC. put shRNA screen after deleting the autophagy cargo receptor and In summary, our results demonstrate for the first time that Etk substrate p62/SQSTM1; PFKFB4 was initially found as an autop- promote the chemoresistance of SCLC by interacting with hagy stimulator in this article; however, the following tests PFKFB4. Furthermore, we identified that knocking down either showed that knockdown of PFKFB4 increased autophagic flux Etk or PFKFB4 significantly reduced autophagic flux while increas- by suppressing the accumulation of reactive oxygen species (ROS) ing chemosensitivity in SCLC. Our study implied that ibrutinib and p62 (36). In SCLC, we identified that knockdown Etk or may synergistically promote chemosensitivity in SCLC cells. PFKFB4 significantly reduced autophagic flux, which suggests Therefore, we conclude that Etk and PFKFB4 are promising that inhibiting autophagy by targeting either Etk or PFKFB4 prognostic biomarkers of the chemotherapy response and that may be a potential strategy in attenuating the chemoresistance inhibiting autophagy by targeting PFKFB4 may be a potential of SCLC patients. Although it is difficult to reconcile the results of strategy for treating patients with drug-resistant SCLC. our study with those of the two discordant studies described above, we believe that the combined use of TEM, CLSM, Disclosure of Potential Conflicts of Interest and Western blotting to monitor autophagy, as well as the No potential conflicts of interest were disclosed. application of siRNAs to downregulate Etk and PRKRB4 in che- Authors' Contributions moresistant SCLC cell lines, provide adequate data to support Conception and design: Q. Wang, F. Zeng, L. Guo our claims that downregulation of Etk or PFKFB4 could signifi- Development of methodology: Q. Wang, Y. Sun cantly inhibit autophagy in SCLC. In addition, the studies men- Acquisition of data (provided animals, acquired and managed patients, tioned above that used prostate cancer cells and non–small cell provided facilities, etc.): F. Zeng, Y. Sun, W. Huang lung cancer cells as the experiment models (19); thus, the dis- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cordant results suggest that in SCLC, PFKFB4 may modulate computational analysis): Q. Wang, F. Zeng, Q. Qiu, X. Huang autophagy by mechanisms other than suppressing either ROS or Writing, review, and/or revision of the manuscript: Q. Wang, F. Zeng, J. Zhang, J. Huang, L. Guo p62 accumulation. Hence, it is critical to elucidate the molecule Administrative, technical, or material support (i.e., reporting or organizing mechanisms by which PFKFB4 contributes to autophagy and data, constructing databases): Q. Wang, F. Zeng, W. Huang present effective regimens to inhibit autophagy for improving Study supervision: L. Guo chemosensitivity in SCLC. Furthermore, to fully understand the relation between Etk and Acknowledgments PFKFB4 in the mRNA and protein level and to find effective The SCLC Human SCLC cell lines NCI-H82, H209, H345, H146 and H526 were obtained as a generous gift from Dr. Ji Lin of MD Anderson Cancer Center. inhibitors targeting Etk and PFKFB4, additional studies are fl fi The ow cytometry analysis was supported by Guangdong Provincial Key required to identify the speci c binding domains of each protein Laboratory of Malignant Tumor Epigenitic and Gene Regulation, Sun Yat-Sen and measure the kinase activity of these two enzymes. Our data Memorial Hospital, Sun Yat-Sen University. This work was supported by grants indicated that PFKFB4 modulated chemoresistance both in vivo from the National Natural Science Foundation of China (nos. 81172241 and and in vitro, and correlated with poor survival and a weak che- 81572244), the Clinical Research Initiative Project of Southern Medical Uni- motherapy response in SCLC. which is consistent with other versity (LC2016ZD029), and Guangdong Natural Science Foundation (Special fi reports that noted PFKFB4 overexpression promotes cancer cell fund for Scienti c and Technological Development; 2017A030313644, 2016A030313822). survival in other cancer types (27–29). Ibrutinib (PCI-32765) is highly effective in controlling some The costs of publication of this article were defrayed in part by the payment of types of B-cell malignancies by inhibiting Btk. On the basis of the page charges. This article must therefore be hereby marked advertisement in effective application of ibrutinib for leukemia patients, an idea accordance with 18 U.S.C. Section 1734 solely to indicate this fact. arose that ibrutinib may help attenuate chemoresistance in SCLC patients because Etk shares similar domains with Btk and Received May 23, 2017; revised October 30, 2017; accepted December 1, Etk is partially inhibited by ibrutinib. Our study revealed that 2017; published OnlineFirst December 5, 2017.

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Wang et al.

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962 Clin Cancer Res; 24(4) February 15, 2018 Clinical Cancer Research

Etk Interaction with PFKFB4 Modulates Chemoresistance of Small-cell Lung Cancer by Regulating Autophagy

Qiongyao Wang, Fanrui Zeng, Yanqin Sun, et al.

Clin Cancer Res 2018;24:950-962. Published OnlineFirst December 5, 2017.

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