Integrin-Linked Kinase (ILK)

Journal of Molecular Histology https://doi.org/10.1007/s10735-020-09888-3

ORIGINAL PAPER

Integrin‑linked kinase (ILK) regulates KRAS, IPP complex and Ras suppressor‑1 (RSU1) promoting lung adenocarcinoma progression and poor survival

Sofa Nikou1 · Marina Arbi2 · Foteinos‑Ioannis D. Dimitrakopoulos3 · Chaido Sirinian3 · Panagiota Chadla1 · Ioanna Pappa1 · Giannoula Ntaliarda4 · Georgios T. Stathopoulos4,5 · Helen Papadaki1 · Vasiliki Zolota6 · Zoi Lygerou2 · Haralabos P. Kalofonos3,7 · Vasiliki Bravou1

Received: 26 April 2020 / Accepted: 13 June 2020 © Springer Nature B.V. 2020

Abstract Integrin-linked kinase (ILK) forms a heterotrimeric protein complex with PINCH and PARVIN (IPP) in Focal Adhesions (FAs) that acts as a signaling platform between the cell and its microenvironment regulating important cancer-related functions. We aimed to elucidate the role of ILK in lung adenocarcinoma (LUADC) focusing on a possible link with KRAS oncogene. We used immunohistochemistry on human tissue samples and KRAS-driven LUADC in mice, analysis of large scale publicly available RNA sequencing data, ILK overexpression and pharmacological inhibition as well as knockdown of KRAS in lung cancer cells. ILK, PINCH1 and PARVB (IPP) proteins are overexpressed in human LUADC and KRAS-driven LUADC in mice representing poor prognostic indicators. Genes implicated in ILK signaling are signifcantly enriched in KRAS-driven LUADC. Silencing of KRAS, as well as, overexpression and pharmacological inhibition of ILK in lung cancer cells provide evidence of a two-way association between ILK and KRAS. Upregulation of PINCH, PARVB and Ras suppressor-1 (RSU1) expression was demonstrated in ILK overexpressing lung cancer cells in addition to a signifcant positive correlation between these factors in tissue samples, while KRAS silencing downregulates IPP and RSU1. Pharmacological inhibition of ILK in KRAS mutant lung cancer cells suppresses cell growth, migration, EMT and increases sensitivity to platinum-based chemotherapy. ILK promotes an aggressive lung cancer phenotype with prognostic and therapeutic value through functions that involve KRAS, IPP complex and RSU1, rendering ILK a promising biomarker and therapeutic target in lung adenocarcinoma.

Keywords ILK · KRAS · Lung cancer · PARVB · PINCH · Ras suppressor-1

Abbreviations ECM Extracellular matrix Electronic supplementary material The online version of this EMT Epithelial to mesenchymal transition article (https://doi.org/10.1007/s10735-020-09888-3) contains ERK1/2 Extracellular regulated kinase supplementary material, which is available to authorized users.

* Vasiliki Bravou 5 Comprehensive Pneumology Center (CPC) and Institute [email protected] for Lung Biology and Disease (iLBD), University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum 1 Department of Anatomy‑Histology‑Embryology, Medical München, Member of the German Center for Lung Research School, University of Patras, 26500 Patras, Greece (DZL), Munich, Bavaria, Germany 2 Department of General Biology, Medical School, University 6 Department of Pathology, University Hospital of Patras, of Patras, 26504 Patras, Greece 26504 Patras, Greece 3 Clinical and Molecular Oncology Laboratory, Division 7 Division of Oncology, Department of Internal Medicine, of Oncology, Medical School, University of Patras, University Hospital of Patras, 26504 Rio, Greece 26504 Rio, Greece 4 Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, 2504 Rio, Achaia, Greece

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FFPE Formalin fxed parafn embedded pathways among which PI3K/AKT is noteworthy, as well FA Focal adhesion as to its role as a scafold protein in FAs (Hannigan et al. GSEA Gene set enrichment analysis 2005). Interestingly, a regulatory ILK-KRAS loop has H-score Histoscore been recently demonstrated in human pancreatic cancer ILK Integrin-linked kinase (Chu et al. 2016) and ILK’s binding partner PINCH has IPP ILK-PINCH-PARVIN been shown to bind Ras suppressor protein 1 (RSU1) IQ-GAP1 Ras-GTPase activating-like protein 1 modulating cell migration and KRAS oncogenic func- LUADC Lung adenocarcinoma tions (Dougherty et al. 2008; Cutler et al. 1992; Elias et al. NSCLC Non small cell lung cancer 2012). These fndings render ILK a promising biomarker OS Overall survival and therapeutic target in KRAS-driven cancers such as qPCR Quantitative PCR lung adenocarcinoma. RSU1 Ras suppressor-1 ILK is overexpressed in several types of cancer includ- WB Western blot ing non-small cell lung cancer (NSCLC) (Zheng et al. 2019; Hannigan et al. 2005; Widmaier et al. 2012). PINCH and parvins overexpression have been also reported in Introduction cancer but their exact role is carcinogenesis is ambiguous (Kovalevich et al. 2011; Xu et al. 2016; Sepulveda and Wu Lung cancer is the main cause of cancer related mortality 2006; Zhang et al. 2004). While PARVA has been shown (Siegel et al. 2019). Lung adenocarcinoma (LUADC), the to promote lung adenocarcinoma metastasis little is known most common histologic type, has shifted away from histo- regarding PINCH1 and PARVB expression in LUADC pathological stratifcation of patients alone towards molecu- (Huang et al. 2015). In the light of recent fndings that IPP lar classifcation regarding alterations in “driver” oncogenes, complex proteins crosstalk with KRAS (Chu et al. 2016; with KRAS oncogene mutations being the most common Dougherty et al. 2008) and the clinical implications that genetic aberration in the disease (Travis et al. 2015; Fer- these interactions may have, we use a series of human lung rer et al. 2018). Molecular genotyping guides treatment of adenocarcinoma samples, bioinformatics tools, in vitro LUADC demonstrating superior efcacy of targeted kinase gain and loss of function experiments and pharmacologi- inhibitors for patients with EGFR mutations or ALK fusions cal inhibition of ILK to decipher ILK’s implication in lung compared to chemotherapy (Chan and Hughes 2015). How- adenocarcinoma progression in relation to KRAS. ever, the patients harboring activating mutations in KRAS do not respond efectively to treatment and little advance has been made in developing clinical efcient KRAS-targeted therapies (Chan and Hughes 2015; Ferrer et al. 2018). In Materials and methods this context identifcation of novel biomarkers and therapeutic targets in KRAS-driven lung adenocarcinoma is a Tissue samples formidable challenge. Focal Adhesions (FAs) serve as signaling platforms Human LUADC tissue samples form 97 patients were between the cell and the extracellular matrix (ECM) retrieved from the Archives of Histopathology, Univer- assuming fundamental roles in the regulation of cell adhe- sity Hospital of Patras. The study was approved by the sion, proliferation and migration processes required for Institutional Ethics & Research Committee according to an tumor growth, invasion and metastasis (Maziveyi and Ala- institutional standardized protocol that abides by the Dec- hari 2017). Integrin-Linked kinase (ILK) is a key regulator laration of Helsinki (protocol number 364/01.08.2017). of FA formation, turnover and signaling (Nikolopoulos Clinicopathological data and disease outcome after a and Turner 2001; Attwell et al. 2003). ILK is located in 60-month observation period were obtained from pathol- FAs where it forms a heterotrimeric complex with PINCH ogy and medical records respectively (Supplementary and parvins, the IPP complex (Legate et al. 2006). IPP Table 1). Histopathology of the tumors was confrmed by proteins PINCH and parvins serve as adaptor proteins due two expert pathologists (VB &VZ) (Travis et al. 2015). to their multiple protein binding motifs with key functions The KRAS G12D/+mouse model of lung adenocarcinomas in the regulation of cell adhesion, cell migration and sur- which conditionally expresses KRAS was developed at vival, while ILK also operates as a kinase in vivo (Legate GT. Stathopoulos Lab as previously described (Agalioti et al. 2006; Fukuda et al. 2009; Wickström et al. 2010). et al. 2017; Johnson et al. 2001). Formalin-fxed parafn ILK has a well-known oncogenic role in cancer (Hannigan embedded (FFPE) lung samples from KRASG12D/+ mice et al. 2005). ILK’s oncogenic properties allocate to activa- were used in this study. tion via its serine-threonine kinase activity of signaling

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Immunohistochemistry gift from Gerhart Ryfel, Addgene plasmid #30530; https:// n2t.net/addgene:30530; RRID _30530) were used as con- Immunohistochemistry was performed on FFPE tissue sam- trol. Briefy ~ 5.5 × 104cells were transfected with a total of ples using a two-step immunoperoxidase method with DAB 1 μg of plasmid DNA using Lipofectamine 2000 (Invitro- as the chromogen (EnVisionTM FLEX Mini Kit High pH, gen, Carlsbad, CA, USA) according to the manufacturer’s K8023, DAKO Carpinteria, CA) as reported previously instructions and analyzed 48 h post transfection. (Tsoumas et al. 2018). Primary antibodies used and appro- priate positive and negative controls are shown in Supple- Quantitative real‑time PCR (qPCR) mentary table 2. Immunohistochemical staining was evaluated by an expert pathologist (VB) blinded to the case, using histoscore (H-score) with the formula: (1 × % cells staining Total RNA from cell lines was isolated using the Nucle- weakly positive) + (2 × % cells staining moderately posi- ospin RNA II kit (MachereyNagel) and 1 μg RNA was tive) + (3 × % cells staining strongly positive) resulting in converted to cDNA using M-MLV reverse transcriptase scores ranging from 0 to 300 as previously described (Tsou- (Takara). mRNA expression levels were assessed by qPCR mas et al. 2018). Images were captured on Nikon Eclipse 80i (Applied Biosystems Step One), using the Kapa SYBR Fast with ACT-1C software (Nikon Instruments Inc., Melville, qPCR kit (KapaBiosystems, KK4605 Wilmington, MA). NY, USA). HPRT or YWHAZ mRNA expression levels were used for normalization. Primer sequences are shown in Supplemen- Cell lines and drugs tary Table 3. No template controls (NTC) were included in each PCR reaction. For qPCR data analysis, the CT values Human lung carcinoma cell lines (A549, H358, H522) were extracted from the Step One cycler (Applied Biosys- used in the study were provided by Dr. Kalofonos H (A549, tems, Carlsbad, CA) and analyzed with the RESTMCS beta H358) and Dr. Lygerou Z (H522) collaborating laborato- software. ries. Cell lines were periodically tested for Mycoplasma Spp. (Mycoprobe CUL001B, R&D Systems). Mouse lung Immunofuorescence adenocarcinoma cell lines CULA (generated from C57BL/6 mice), FULA (generated from FVB mice) and FULA with Cells were fxed with 4% paraformaldehyde for 10 min and stable knockdown of KRAS by shRNA (FULAshKRAS) immunofuorescence was then performed as described pre- were generated in Stathopoulos GT laboratory as reported viously (Tsoumas et al. 2018). Primary antibodies used are (Agalioti et al. 2017). H522 and CULA cell lines are wild shown in Supplementary Table 2. Alexa Fluor-labeled sec- type in respect to KRAS, while A549, H358 and FULA bear ondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG, KRAS mutations. All cell lines were cultured at 37 °C in 5% Alexa Fluor 641 goat anti-mouse and Alexa Fluor 568 goat CO 2- 95% air using full culture medium (DMEM supple- anti-mouse IgG, Invitrogen, Thermo Fisher Scientifc, USA) mented with 10% FBS (Gibco), 1 mM pyruvate, 100 U/ ml were used at 1:700 diluted in blocking solution, for 90 min. penicillin and 100 mg /ml streptomycin). The specifc ILK DNA was stained with Hoechst 33258 (1:1000, Sigma). Sec- inhibitor QLT0267 was purchased from Dermira (Menlo tions were mounted in Mowiol 4–88 (Calbiochem). Quanti- Park, CA, USA) and was diluted in DMSO. For pharmaco- fcations were carried out on flters from at least three inde- logical ILK inhibition or pretreatment with QLT0267, cells pendent experiments. Images from immunofuorescence were treated with QLTO267 at concentration equal to IC50 were recorded on confocal fuorescence microscopy Leica (IC50A549 = 13μΜ ΙC50H522 = 20μΜ ΙC50H358 = 20μΜ) SP5 using 40 × and 63 × lenses and in an inverted Nikon estimated by MTT assay or DMSO (control) for 12 h. Cis- Eclipse TE 200‐U microscope using 20 × and 40 × lenses. platin was diluted in water for injection (1 mg/ml). For EGF Digital images were processed with Fiji software. stimulation cells (A549 or A549 pretreated with QLT0267) were treated with EGF (PeproTech Inc, 100 ng/ml) for 10 min. Immunoblotting

Transient transfection Cells were lysed in lysis bufer (10 mM Tris–HCl pH 8, 140 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton, 1 mM ILK overexpression was established by transient transfec- sodium fuoride, 0.1% sodium deoxycholate, 0.5 mM Na- tion of H522 cells with tdTomato-ILK-N-17 plasmid (a gift orthovanadate and 1% protease Inhibitor). Immunoblotting from Michael Davidson, Add gene plasmid # 58104; https:// was performed as previously described (Tsoumas et al. n2t.net/addgene:58104 ; RRID:Addgene_58104). H522 cells 2018). Primary antibodies are shown in Supplementary transfected with the empty pCSCMV: tdTomato vector (a Table 2.

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MTT assay and soft agar‑assay Statistical analysis

Drug sensitivity and cell proliferation was evaluated by Statistical analysis was performed using the Statistical 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium Package for Social Sciences version 25 (SPSS, Chicago, IL, bromide (MTT) assay as previously described (Tsoumas USA). Diferences between groups were tested with non- et al. 2018). To evaluate drug sensitivity cell lines were parametric tests (Kruskal–Wallis for k or the Mann–Whitney incubated with QLT0267 for 12 h at a concentration range for 2 independent samples). Correlations were assessed with 0–120 μM or cisplatin for 48 h at 0- 250 μM and IC50 the Spearman’s correlation test. Survival analysis was per- values (the half maximal inhibitory concentration of cell formed with Kaplan Meier and diferences between groups viability or cell metabolism) were calculated. were calculated with the log-rank test. Best cut-of points, For soft agar colony formation assay 7.5 × 103 A549 provided by X-tile software, were used to separate cases into cells pretreated with QLT0267 or DMSO (control) were low and high expression cohorts (Camp et al. 2004) Uni- plated onto 6-well plates in semi-solid 0.7% agarose in full variate and multivariate analysis was performed using Cox’s culture medium and were incubated for 20 days at 37 °C in proportional hazards model. Statistical analysis of in vitro a 5% CO2 humidifed incubator. 2 ml fresh culture medium data (pair-wise comparisons) was performed with Graph- was added to each well biweekly. After incubation, plates Pad Prism version 7.00 (GraphPad Software, La Jolla Cali- were photographed in an inverted microscope and colonies fornia USA) using an unpaired, two-sided Student’s t-test were counted. and non-linear regression. p-values < 0.05 were considered signifcant.

Wound healing assay Results Cells were seeded in six-well plates at 80–90% confu- ency. The cell monolayer was mechanically scraped with ILK, PINCH1 and PARVB proteins are overexpressed a sterile 200-μl pipette tip after complete medium removal in human lung adenocarcinomas and associate and immediately afterwards, cell debris was removed and with low overall survival medium was renewed. The wound closure was monitored for 24 h and photos were taken under an inverted micro- Protein expression of ILK, PINCH1 and PARVB was scope. Results are expressed as % wound area (cell-free examined by immunohistochemistry in 97 cases of human area) at a given time (12, 24 h) relative to the original LUADC. ILK is overexpressed in LUADC with cytoplas- wound area (0 h). mic staining in 86/97 (88.66%) and nuclear staining in 65/97 (32.99%) of cases with mean H-scores 71 ± 6.39 and 25 ± 3.95 respectively. PINCH1 and PARVB are also overex- Bioinformatics analysis pressed in LUADC with cytoplasmic and nuclear localization in tumor cells. Cytoplasmic and nuclear PINCH1 immuno- The Km plotter was used for overall survival (OS) analysis reactivity is observed in 81/97 (83.51%) and 39/97 (40.21%) of mRNA gene chip data (from GEO, EGA, and TCGA) of cases with mean H-scores 72 ± 6.54 and 16 ± 3.45 respec- from 720 LUADC patients (Nagy et al. 2018). Gene Set tively. PARVB cytoplasmic and nuclear immunostaining is Enrichment Analysis (GSEA) [27]) (https://www.broad found in 84/97 (86.66%) and 33/97 (34.02%) of cases with institute.org/gsea/index.jsp) was performed on level 3 mean H-scores 62 ± 6.53 and 9 ± 2.15 respectively. (Fig. 1a). RNAseq TCGA-LUADC dataset (n = 523) from The Can- PINCH1 cytoplasmic expression is signifcantly higher in cer Genome Atlas (TCGA) using the v4.0.3 software (Sub- advanced stage disease (p = 0.036) while both cytoplasmic ramanian et al. 2005). Two set of genes were analyzed: a and nuclear expression of PARVB associate with advanced set of 53 genes of the ILK signaling pathway (Schaefer disease stage (p = 0.01 and p = 0.009 respectively) and lymph et al. 2009) and a second set of 7 genes including only node metastasis (p = 0.037 and p = 0.043 respectively). IPP and RSU1 (Supp. Table 4). The mean of the genes Kaplan–Meier analysis shows a statistically signifcant was calculated with the Signal2Noise statistical score. A association of high cytoplasmic ILK, and PARVB with p-value ≤ 0.05 was considered statistically signifcant. Co- reduced 5-year and high cytoplasmic PINCH with reduced expression and enrichment analysis was also performed 3-year Overall Survival (OS) (38 months for high ILK while on RNA-Seq TCGA dataset using cBioPortal for Cancer it did not reach the median for low ILK, Log-rank p = 0.024, Genomics (https://www.cbioportal.org) in 510 LUADC 30 vs 22 months for low PINCH1, Log-rank p = 0. 03, 31 patients (Gao et al. 2013; Cerami et al. 2012). vs 52 months for low PARVB, Log-rank p = 0.044). Univari- ate analysis shows that pT (pathologic Tumor status), pN

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Fig. 1 Overexpression of ILK, PINCH1 and PARVB in lung adeno- three proteins in tumors compared to adjacent non-neoplastic epithe- carcinoma correlates with poor prognosis. a Immunohistochemi- lium. Bars correspond to 30 μm b Kaplan Meier plots showing asso- cal staining of ILK, PINCH1 and PARVB in representative cases of ciation of high ILK, PINCH1 and PARVB expression with reduced human lung adenocarcinoma (× 400) and in KRAS G12D + -driven overall survival. Log-rank p values < 0.05 are considered statistical mouse model of lung cancer (× 200) showing overexpression of all signifcant LUADC lung adenocarcinoma 1 3

Journal of Molecular Histology ◂ (pathologic Node status), stage, ILK (5-year OS p = 0.03), Fig. 2 ILK-KRAS cross talk in lung adenocarcinoma. a Enrichment PINCH1 (3-year OS p = 0.038) and PARVB (5-year OS plots from GSEA showing signifcant enrichment of ILK pathway gene set (n = 53 genes, left) and IPP pathway gene set (n = 7) includ- p = 0.049) expression are signifcant predictors of poor overall ing IPP components and RSU1 (right) in mutant KRAS lung adeno- survival (Supplementary Table 5). Moreover, in multivariate carcinoma (TCGA, n = 523). b mRNA expression of ILK, PINCH1, analysis high ILK cytoplasmic expression is an independ- PARVB and RSU-1 by qPCR in wild type (H522) and mutant ent predictor of poor OS (Table 1). Results were validated (A549) KRAS lung cancer cells. c Efects of stable KRAS knockdown (shKRAS) on the mRNA expression of ILK, PINCH1, PARVB by Kaplan Meier plotter. Meta-analysis performed on 720 and RSU-1 by qPCR in FULA cells. d Efects of ILK overexpres- LUADC patients shows that high ILK and PARVB mRNA sion (TdtomatoILK) on KRAS mRNA expression by qPCR in H522 levels are associated with poor OS in LUADC (Supplemen- cells. Control H522 cells transfected with empty vector, TdtomatoILK tary Fig. 1). H522 ILK overexpressing cells. e Efects of pharmacologic inhibition of ILK by QLT0267 on KRAS mRNA expression by qPCR in Overexpression of IPP proteins is also demonstrated A549 cells. A549Q + A549 cells treated with QLT0267, Control by immunohistochemistry in lung tumors and preinvasive A549 treated with DMSO. Values correspond to the expression lev- lesions of mice in the KRASG12D/+animal model (n = 5 mice els (mean ± SEM, n = 3) of each gene relative to control values set as with paired normal tissue) (Fig. 1a). 1 following normalization against mRNA levels of reference genes. YWHAZ and HPRT were used as reference genes in human (A549, H522) and mouse (FULA, FULAshKRAS) lung cancer cell lines Evidence of ILK ‑KRAS crosstalk in lung respectively. qPCR data from three independent experiments were adenocarcinoma analyzed by the REST‐MCS beta software. Statistical analysis was performed with Student’s t-test. *p < 0.05; *p < 0.01; ***p < 0.001. SEM standard error of the mean Genes set enrichment analysis revealed that a set of 53 genes of the ILK pathway, as well as, a set of 7 genes including only IPP components and RSU1 are signifcantly enriched in To obtain a mechanistic rationale of ILK-KRAS crosstalk KRAS mutant compared to non-KRAS mutant lung adeno- in lung adenocarcinoma we frst examined whether KRAS carcinomas (n = 523) (Fig. 2a, Supplementary Tables 4 and 6). afects ILK expression in lung cancer cells. Increased levels of ILK are observed in KRAS mutant A549 cells ver- sus wild-type KRAS H522. RSU1 mRNA is also higher in Table 1 Multivariate Cox regression analysis for overall survival KRAS mutant cells while no statistically signifcant differences are observed for PINCH1 and PARVB (Fig. 2b). Multivariate analysis Overall survival Silencing of KRAS in FULA cells signifcantly lowers lev- HR 95% CI p-value els of ILK both in the mRNA level as shown by qPCR and Timepoint 2 years the protein levels (Fig. 2c and immunofuorescence data not pT 2.018 1.131–3.600 0.017 shown). Interestingly, knockdown of KRAS also downregu- pN 1.426 0.597–3.405 0.424 lates expression of the other IPP components PINCH1 and Stage 1.137 0.506–2.558 0.756 PARVB, as well as, RSU1 in lung cancer cells (Fig. 2c). PARVB cytoplasmic Low/High 0.388 0.128–1.177 0.095 We then tested the efect of ILK on KRAS. ILK overex- Timepoint 3 years pression in H522 cells results in increased KRAS mRNA pT 1.007 0.509–1.990 0.985 levels by qPCR analysis (Fig. 2d), while pharmacological pN 1.134 0.471–2.732 0.780 inhibition of ILK with QLT0267 downregulates KRAS Stage 0.919 0.385–2.193 0.849 mRNA (Fig. 2e). PINCH1 cytoplasmic Low/High 1.102 0.338–3.593 0.872 PARVB cytoplasmic Low/High 0.990 0.495–1.978 0.976 ILK regulates PARVB, PINCH and RSU1 expression Timepoint 5 years but not p‑ERK1/2 pT 1.602 0.978–2.625 0.061 pN 1.325 0.71–2.471 0.376 Stage 0.991 0.543–1.806 0.975 To further get insight into the mechanisms involved in ILK cytoplasmic Low/High 0.391 0.171–0.892 0.026 ILK-KRAS crosstalk we analyzed RNA sequencing data PARVB cytoplasmic Low/High 0.634 0.322–1.246 0.186 (n = 510) form the TCGA study using cBioportal online tool. While a direct association between ILK and KRAS mRNA All factors were evaluated in relation to 2-year, 3-year and 5-year expression is not verifed, several factors involved in KRAS overall survival signaling are among the most signifcantly co-expressed Signifcant p-values appear in italics. Only parameters that showed genes with ILK (RRAS, Spearman r = 0.494, p = 9.65e−33) signifcant diference by univariate analysis were included in the multivariate analysis or among the top 10 altered genes in samples with ILK over- HR hazards ratio, CI confdence interval, pT pathologic tumor status, expression (RRAGC, p = 4.52e−9 m q = 2.028e−5) (data not pN pathologic node status, Stage according to AJCC 8th edition shown). Interestingly among the factors co-expressed with

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Journal of Molecular Histology ◂ ILK are PINCH1 (Spearman r = 0.19 p = 1.163e−5), PARVB Fig. 3 ILK regulates the levels of PARVB, PINCH1 and RSU1 in (r = 0.13 p = 3.154e−3) and RSU1 (r = 0.27 p = 4.51e−10) lung adenocarcinoma. a Efects of ILK overexpression on mRNA levels of IPP and RSU1 in H522 cells by qPCR. Control H522 (Supplementary Fig. 2). cells transfected with empty vector, TdtomatoILK H522 ILK over- We also show, by qPCR, that ILK overexpression expressing cells. b Double immunofuorescence analysis of ILK upregulates expression of PINCH1, PARVB and RSU-1 (red)-PINCH1 (grey), ILK (red)-PARVB (green) and ILK (red)- (Fig. 3a). ILK colocalizes with PINCH1, PARVB and RSU1 (green) showing increased numbers of double positive cells (merge) in ILK overexpressing cells (TdTomatoILK) compared to RSU1, mainly in the cell cytoplasm, as shown by immuno- control (H522 transfected with empty vector). Inserts (upper right) fuorescence and ILK overexpression increases the number show higher magnifcation of areas boxed with dashed lines. Arrows of double positive ILK-PINCH1, ILK-PARVB and ILK- indicate double positive cells. Scale bars: 50 μm. Bar graphs depict RSU1 cells (Fig. 3b). We then determined the efect of the percentage of double positive cells compared to ILK positive cells from at least three independent experiments. c Efects of pharmaco- ILK inhibition on IPP complex and RSU1 levels, using logic inhibition of ILK with QLT0267 on the mRNA levels of ILK, QLT0267. QLT0267 reduces ILK, PINCH1 and RSU1 PINCH1, PARVB and RSU1 by qPCR in A549 cells. A549Q+ A549 both at the mRNA and protein levels, by qPCR (Fig. 3c) cells treated with QLT0267, Control A549 treated with DMSO. For and immunofuorescence (Supplementary Fig. 2) respec- qPCR, values correspond to the expression levels (mean ± SEM from three replicates) of each gene relative to control values set as tively, while PARVB levels fuctuate. In human tissue 1 following normalization against YWHAZ mRNA levels (reference samples examined by immunohistochemistry ILK expres- gene). qPCR data were analyzed by the REST‐MCS beta software. sion correlates signifcantly with expression of PINCH1 Statistical analysis was performed with Student’s t-test. *p < 0.05; (Spearman r = 0.342 p = 0.001) and RSU1 (Spearman **p < 0.01; ***p < 0.001. SEM standard error of the mean. (Color fg- ure online) r = 0.275 p = 0.007). As extracellular regulated kinase ERK1/2 is a key efector of the RAS pathway we next evaluated relation (ΙC50H522 = 20.37 ± 0.56 vs IC50A549 = 13.31 ± 3.34, of ERK1/2 to ILK signaling in lung cancer (Ferrer et al. p = 0.1 and IC50CULA = 25.57 ± 6.9 vs 2018). p-ERK1/2 expression was evaluated by immuno- IC50FULA = 14.78 ± 0.22, p = 0.19). However, QLT0267 histochemistry in lung adenocarcinoma samples. Positive more efciently suppresses growth of FULA cells com- cytoplasmic and nuclear p-ERK1/2 expression is found pared to FULAshKRAS as IC50 values for FULAshKRAS in 25/97 (25.77%) and in 56/97 (57.73%) of cases with is 93.36 ± 6.67 compared to 25.57 ± 6.92 for FULA mean H-scores 13 ± 3.66 and 25 ± 4.11 respectively. High (p = 0.001) (Fig. 4a). nuclear p-ERK expression is associated with reduced Pharmacological inhibition of ILK also reduces anchor- 2-year OS by Kaplan Meier analysis (log-rank p = 0.001). age independent growth of A549 cells by soft agar colony However, there is no significant association between formation assay as QLT0267 signifcantly reduces number expression of p-ERK1/2 and ILK, PINCH1 or PARVB in of colonies formed after 20 days (Fig. 4b). tissues samples. Levels of p-ERK were also evaluated by QLT0267 also hampers wound closure at the cell mon- western immunoblotting in A549 cells exposed to EGF. olayer and decreases cell migration leading to signifcantly Pharmacological inhibition of ILK with QLT0267 does wider cell free area after 24 h observation in A549 cells not alter p-ERK levels in lung cancer cells in response to treated with QLT0267 compared to control as shown by EGF (Supplementary Fig. 3). wound healing assay (Fig. 4c).

Pharmacologic inhibition of ILK reduces cell ILK regulates EMT in lung adenocarcinoma proliferation, anchorage‑independent growth and migration of lung cancer cells Previous studies have underlined an important role of ILK in EMT and evidence from pancreatic cancer also impli- Preclinical studies have assessed the use of specifc ILK cate ILK in KRAS induced EMT a process that is funda- inhibitor QLT0267 for cancer therapy with promising mental for cancer invasion and metastasis (Hannigan et al. results (Edwards et al. 2005; Dos Santos et al. 2006). We 2005; Chu et al. 2016). We frst examined whether EMT here show that pharmacological inhibition of ILK with is implicated in the oncogenic functions of ILK. Upon QLT0267 efectively inhibits cell growth rates of human ILK overexpression, EMT markers ZEB1 and Vimen- and mouse lung cancer cell lines as shown by MTT assay. tin are overexpressed, while E-cadherin is downregulated Although IC50 values of QLT0267 are lower in mutant as shown by qPCR (Fig. 5a). We also evaluated ZEB and KRAS compared to wild-type KRAS lung cancer cell E-cadherin expression in our cohort of tumors by immuno- lines differences do not reach statistical significance histochemistry. ZEB nuclear expression is observed in 60/97

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◂Fig. 4 Pharmacologic inhibition of ILK with QLT0267 reduces We here provide novel evidence that ILK regulates KRAS, cell proliferation, anchorage-independent growth and migration IPP and RSU1 promoting lung cancer progression and poor of lung cancer cells in vitro. a Assessment of cell growth and proliferation rates in human (A549 and H522) and mouse (CULA, patient survival, rendering ILK a promising therapeutic tar- FULA, FULAshKRAS) lung cancer cell lines by MTT assay follow- get in lung adenocarcinoma. ing treatment with the ILK inhibitor QLT0267. Logarithmic curves In our study, ILK is overexpressed in human lung adeno- depict cell growth rates and bars show comparison of IC50 values carcinoma and represents an independent poor prognostic (mean of 3 replicates ± SD) between cells lines. b A549 cells were grown in soft agar in the presence of DMSO (control) or QLT0267 indicator, supporting an important role of ILK in lung adeno- (A549Q+) (× 100). Scale bars at 50 μm. Data (total colony count carcinoma progression. In accordance, ILK is a well-estab- expressed as means) from three independent experiments is shown. lished oncogene and overexpression has been previously c In vitro scratch wound healing assay in A549 cells upon treatment reported in several cancer types, including LUADC (Zheng with QLT0267 (A549Q+) compared to control (A549 cells treated with DMSO). Representative micrographs (of three independent et al. 2019; Hannigan et al. 2005; Takanami 2005) In further experiments) show the original wound and the wound after 12 and agreement with a role of ILK in lung adenocarcinoma pro- 24 h. Quantifcation was performed by Image J. The wound area was gression, overexpression of ILK has been previously shown determined as the percentage of wound area (cell -free area) at 24 h to induce lung cancer cell migration and invasion via NF- relative to the original wound area (0 h) Diferences were determined by two-sample Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001 kappa-mediated upregulation of matrix metalloproteinase-9 (Zhao et al. 2013). Additionally, increased expression of ILK and α-catulin have been associated with poor prognosis in (61.86%) cases with mean H-score 23 ± 3.85 and E-cadherin NSCLC and promote an invasive phenotype by activating shows membranous, cytoplasmic and nuclear staining in an ILK-mediated Akt-NF-kB-αvβ3 axis (Liang et al. 2013). 67/97 cases (69.07%) with mean H-score 64 ± 8.59, 48/97 Although PARVA has been previously shown to promote (49.98%) cases with mean H-score 37 ± 6.55 and in 28/97 lung cancer migration and metastasis, little is known for (68. 04%) cases with mean H-score 10 ± 2.41 respectively ILK’s binding partners PINCH1 and PARVB in lung cancer (Fig. 5b). ILK as well as PARVB and PINCH1 protein lev- (Huang et al. 2015). Here, we report for the frst time that els correlate with EMT markers in our cohort of tumors PINCH1 and PARVB are overexpressed in human lung ade- (ILK-ZEB Spearman r = 0.298 p = 0.002, ILK-cytoplasmic nocarcinoma and correlate with short overall survival, sug- E-cadherin r = 0.282 p = 0.006, PARVB-ZEB1 r = 0.253 gesting that ILK’s binding partners in FAs contribute to an p = 0,019, PINCH1-ZEB1 r = 0.218 p = 0.042) Moreover, aggressive lung cancer phenotype. PINCH and parvins act pharmacological inhibition of ILK with QLT0267 efciently as a protein scafold at FAs where through adaptor functions reduces ZEB1 and Vimentin mRNA (Fig. 5c) and protein they regulate focal adhesion assembly, cell adhesion, migra- levels (data not shown) but it does not restore E-cadherin tion and survival (Legate et al. 2006; Sepulveda and Wu levels (Fig. 5c). QLT0267 also induces phenotypic changes 2006). PINCH1 interestingly cross talks with growth factor consistent with reversal of an EMT phenotype in mutant receptors through interaction with NCK2 (Xu et al. 2016) KRAS A549 cells (Fig. 5d). and, as recently shown, with KRAS signaling through RSU1 (Dougherty et al. 2008; Cutler et al. 1992). In agreement Pharmacological inhibition of ILK increases with our fndings, a role of PINCH1 and PARVB in human sensitivity to cisplatin in human KRAS mutant lung cancer has been previously evidenced (Tsinias et al. 2018; cancer cells Bravou et al. 2015; Lööf et al. 2011). Increased expression of PINCH1 has been reported in the invasive front of several We then tested whether QLT0267 sensitizes KRAS mutant tumors (Wang-Rodriguez et al. 2002) and levels of PINCH lung cancer cells to platinum-based therapy. A549 and H358 have been associated with poor prognosis in pancreatic and cells were treated for 48 h with cisplatin alone (control) or laryngeal cancer (Huang et al. 2019; Tsinias et al. 2018) cisplatin after pretreatment with QLT0267. QLT0267 sig- However, PARVB exerts a more controversial, tissue specifc nifcantly increases sensitivity to cisplatin as shown by loga- role in cancer, as both tumor suppressive and tumor pro- rithmic growth curves and IC50 values comparison (Fig. 5e). moting roles for PARVB have been described (Eslami et al. 2014; Bravou et al. 2015; Mongroo et al. 2004; Wu et al. 2010) Nevertheless, evidence coming mainly from studies Discussion in laryngeal carcinoma and colon cancer (Tsinias et al. 2018; Bravou et al. 2015) support our fndings of a tumor promot- Understanding mechanisms underlying lung cancer progres- ing role of PARVB in human lung adenocarcinoma. sion and discovery of novel biomarkers and therapeutic tar- Signifcantly, we provided evidence supporting the notion gets are of outmost importance, especially for KRAS-driven that ILK cross talks with KRAS in lung adenocarcinoma. lung adenocarcinoma, where clinical efective therapies are In line with our fndings proteomic studies have shown that still lacking (Ferrer et al. 2018; Chan and Hughes 2015). ILK interacts with RAS-GTPase activating-like protein 1

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(IQ-GAP1) which activates the RAS pathway (Fielding et al. Furthermore, ILK promotes cell migration through activa- 2011). ILK also induces activation of the RAS/ERK1/2/ tion of small GTPases RAC and CDC42, that represent sig- NF-κB axis in gastric cancer cells (Tseng et al. 2014) nifcant efectors of RAS signaling (Filipenko et al. 2005).

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◂Fig. 5 ILK regulates EMT and sensitivity to cisplatin in lung adeno- central component of an interactome protein network in FAs carcinoma. a Overexpression of ILK in H522 cells (TdtomatoILK) modulating signaling pathways through protein interactions induces EMT as it upregulates mRNA expression of ZEB1 and Vimentin and downregulates E-cadherin, as shown by qPCR analy- (Fukuda et al. 2009; Wickström et al. 2010) For instance, sis. b EMT markers are expressed in human lung adenocarcinoma. ILK binds to PARVA and PARVB and induces assembly of Representative immunohistochemical stains of E-cadherin and ZEB1 AKT into the plasma membrane, where it exerts its onco- in human lung adenocarcinoma showing cytoplasmic and nuclear genic activity (Attwell et al. 2003).Overall these evidence E-cadherin in tumor cells compared to membranous staining in non- support the notion that interactions of ILK with IPP complex neoplastic epithelium and overexpression of ZEB. c mRNA expression of ZEB, Vimentin and E-cadherin by qPCR upon pharmacologic in FAs may be important for ILK oncogenic role in lung inhibition of ILK with QLT0267 in A549 cells. A549Q+ A549 cells adenocarcinoma (Widmaier et al. 2012; Maziveyi and Ala- treated with QLT0267, Control A549 cells treated with DMSO. d hari 2017; McDonald et al. 2008). QLT0267 reverses EMT related morphological changes in A549 cells, as spindle cells lose elongated protrusions and acquire a Co-expression of ILK with RSU1 in human tissue sam- rounded shape (magnifcation 100 ×). Bar at 50 μm. A549Q + A549 ples and upregulation of RSU1 in ILK overexpressing cells cells treated with QLT0267, Control A549 cells treated with DMSO implicate RSU1 in ILK oncogenic functions in LUADC. e QLT0267 sensitizes KRAS mutant lung cancer cells (A549, H358) RSU1 was frstly identifed by its ability to inhibit RAS to platinum-based therapy. MTT assay assessing cisplatin sensitivity in A549 and H358 cells pretreated or not with QLT0267. Logarithmic dependent transformation and next it was shown to interact curves (left) show cell growth rates and bars (right) show comparison with the IPP complex through PINCH in FAs, regulating cell of IC50 values (mean of 3 replicates ± SD of cisplatin between cells migration, invasion and metastasis in many types of cancers lines). A549Q+ A549 cells pretreated with QLT, H358Q+ Η358 cells (Cutler et al. 1992; Zacharia et al. 2017; Dougherty et al. pretreated with QLT. For qPCR analysis values correspond to the expression levels (mean ± SEM from three replicates) of each gene 2008). Evidence supporting critical involvement of RSU1 relative to control values set as 1 following normalization against in ILK signaling also comes for studies of neural stem and YWHAZ mRNA levels. qPCR data were analyzed by the REST‐MCS progenitor cell proliferation in adult brain (Porcheri et al. beta software. Statistical analysis was performed with Student’s t-test. 2014). However, the role RSU1 exerts in lung adenocarci- *p < 0.05; **p < 0.01; ***p < 0.001. SEM standard error of the mean noma is not known and addressing this question in future studies is of priority. In further accordance, it has been previously shown that ILK Despite previous data linking ILK to RAS/ERK1/2 sign- upregulates KRAS expression and KRAS in turn upregulates aling (Noguchi et al. 2013; Tseng et al. 2014) we provided ILK expression in pancreatic cancer cells and this regula- evidence that ERK is not involved in ILK-KRAS cross talk. tory feedback loop enhances the oncogenic actions of KRAS On the other hand, IPP proteins and RSU1 are enriched in pancreatic cancer (Chu et al. 2016). However, there is in KRAS mutant lung adenocarcinomas and silencing of also a single study that failed to show ILK-KRAS cross- KRAS in lung cancer cells not only suppressed levels of talk in lung and colon cancer (Chu et al. 2018). Considering ILK but also levels of PINCH1 PARVB and RSU1. Based that a stochastic approach for targeting oncogenic KRAS on these parallel functions we speculate that regulation of is through upstream signaling these fndings are of clinical PINCH1, PARVB and RSU1 may be involved in ILK-KRAS importance as ILK may represent a novel therapeutic target crosstalk in lung adenocarcinoma. In support of this, RAS in lung adenocarcinoma. signaling alters RSU1 splicing and interaction with PINCH We also showed that PINCH1 and PARVB are co- in FAs modulating cell migration (Dougherty et al. 2008; expressed with ILK and ILK overexpression upregulates Gonzalez-Nieves et al. 2013). However, a mechanistic PINCH1 and PARVB expression and increases their co- rationale of this regulatory loop, if indeed exists, between localization with ILK in lung cancer cells. These fndings ILK-KRAS-IPP-RSU1 needs to be explored. suggest that PINCH and PARVB are implicated in ILK’s To further support a clinically relevant role of ILK oncogenic functions in lung adenocarcinoma. In accordance, in lung adenocarcinoma we showed that pharmacologic association between ILK and IPP components expression inhibition of ILK with QLT0267, not only downregulates has been previously reported in colorectal cancer (Bravou KRAS, PINCH1 and RSU1 expression, but also sup- et al. 2015) and depletion of ILK has been also shown to press proliferation and anchorage independent growth of result in downregulation of parvins and PINCH in previ- lung cancer cells in vitro. Promising anti-cancer efects ous studies (Stanchi et al. 2009; Fukuda et al. 2003). An of QLT0267 have been previously documented in other important role of IPP in cell-ECM interactions and sign- cancers (Edwards et al. 2005; Dos Santos et al. 2006). In aling has already been emphasized (Legate et al. 2006). agreement with our fndings another ILK inhibitor (KP- Although activation of PI3K/AKT and β-catenin pathways 392) showed anti-tumor efcacy both in vitro and in an attributable to kinase activity of ILK have been linked to orthotopic animal model (Liu et al. 2006). In addition, a ΙLK mediated carcinogenesis in several studies (Hannigan noteworthy observation was that levels of KRAS afect et al. 2005; Bravou et al. 2006) recent fndings dispute over sensitivity of lung cancer cells to growth inhibition by ILK the "kinase" function of ILK, suggesting that ILK acts as a

1 3 Journal of Molecular Histology inhibitor QLT0267 further supporting an ILK-KRAS cross Ethical approval The study was approved by the Institutional Ethics talk in lung adenocarcinoma. & Research Committee of the University Hospital of Patras (protocol number 364/01.08.2017). Since archival tissues were used in the study Furthermore, we demonstrated that ILK overexpression the informed consent process was waived. Mice were bred at the Center induces EMT while pharmacological inhibition of ILK for Animal Models of Disease of the Department of Physiology at suppresses cell migration and EMT in lung cancer cells. the Faculty of Medicine of University of Patras, Greece. The animal These fndings suggest that EMT mechanisms are critically study was approved by the Veterinary Administration of the Prefecture of Western Greece (#118018/578/30.04.2014) and were conducted in involved in ILK oncogenic functions in lung adenocarci- accordance to Directive 2010/63/EU (http://eurlex.europa.eu/legal noma. In line with this an important role of ILK in inducing -content/EN/TXT/?uri=CELEX%3A32010L0063) as described by EMT and thus promoting cancer invasion and metastasis Agalioti et al. 2017. has been previously demonstrated in human cancers, including lung adenocarcinoma (Chen et al. 2013; Tsoumas et al. 2018; Bravou et al. 2006; Fuchs et al. 2008) Interestingly References ILK has been shown to suppress RAS-mediated EMT in pancreatic cancer (Chu et al. 2016). Whether this is also the Agalioti T, Giannou AD, Krontira AC, Kanellakis NI, Kati D, Vreka case for lung adenocarcinoma needs further research. M, Pepe M et al (2017) Mutant KRAS promotes malignant Also, inhibition of ILK increases sensitivity of KRAS pleural efusion formation. Nat Commun 8:15205. https://doi. mutant lung cancer cells to cisplatin. In agreement, synergy org/10.1038/ncomms15205 between ILK targeting and classical chemotherapeutics Attwell S, Mills J, Troussard A, Wu C, Dedhar S (2003) Integration of cell attachment, cytoskeletal localization, and signaling by inte- has been previously evidenced in lung cancer cell in vitro grin-linked kinase (ILK), CH-ILKBP, and the tumor suppressor (Zhao et al. 2015; Jia 2015).Previous reports also support an PTEN. Mol Biol Cell 14(12):4813–4825. https://doi.org/10.1091/ important role of ILK in cancer chemoresistance (Duxbury mbc.e03-05-0308 et al. 2005; Tsoumas et al. 2018). In colorectal cancer for Bravou V, Klironomos G, Papadaki E, Taraviras S, Varakis J (2006) ILK over-expression in human colon cancer progression corre- instance we have previously showed that inhibition of ILK lates with activation of β-catenin, down-regulation of E-cadherin reduces chemoresistance through EMT-related mechanisms and activation of the Akt–FKHR pathway. J Pathol 208(1):91–99. (Tsoumas et al. 2018). Overall, these fndings render anti- https://doi.org/10.1002/path.1860 ILK targeting alone or in combination with chemotherapy Camp RL, Dolled-Filhart M, Rimm DL (2004) X-tile. Clin Cancer Res 10(21):7252–7259. https://doi.org/10.1158/1078-0432. very promising in lung adenocarcinoma. CCR-04-0713 In conclusion our study delineates the important, clini- Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, Jacob- cally relevant role of ILK in lung adenocarcinoma. We also sen A et al (2012) The CBio cancer genomics portal: an open provide a mechanistic rationale for ILK oncogenic functions platform for exploring multidimensional cancer genomics data. Cancer Discov 2(5):401–404. https://doi.org/10.1158/2159-8290. implicating ILK-KRAS cross-talk and IPP-RSU1 in ILK- CD-12-0095 mediated lung carcinogenesis. Future research is warranted Chan BA, Hughes BG (2015) Targeted therapy for non-small cell regarding the implication of FA proteins in ILK-KRAS inter- lung cancer: current standards and the promise of the future. action and the role of RSU1 in lung adenocarcinoma. Transl Lung Cancer Res 4(1):36–54. https://doi.org/10.3978/j. issn.2218-6751.2014.05.01 Chen D, Zhang Y, Zhang X, Li J, Han B, Liu S, Wang L, Ling Y, Mao Acknowledgments We thank the Advanced Light Microscopy Facility, S, Wang X (2013) Overexpression of integrin-linked kinase cor- University of Patras, for help with microscopy. relates with malignant phenotype in non-small cell lung cancer and promotes lung cancer cell invasion and migration via regu- Author contributions SN: Investigation, Visualization, Writing of orig- lating epithelial-mesenchymal transition (EMT)-related genes. inal draft; MA, CS, PC, IP, GN: Investigation; FDD: Formal analysis, Acta Histochem 115(2):128–136. https://doi.org/10.1016/j.acthi GTS, HP, VZ, ZL, HPK: resources, writing, reviewing and editing; VB: s.2012.05.004 Conceptualization, Methodology, Funding acquisition, Supervision, Chu P-C, Yang M-C, Kulp SK, Salunke SB, Himmel LE, Fang C-S, Visualization, Writing of original draft, reviewing &editing. Jadhav AM et al (2016) Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-HnRNPA1 regulatory Funding This work was supported by the Basic Research Grant "K. loop in human pancreatic cancer cells. Oncogene 35(30):3897– Karatheodori" (E658) of the University of Patras (to Vasiliki Bravou). 3908. https://doi.org/10.1038/onc.2015.458 Chu PC, Kulp SK, Bekaii-Saab T, Chen CS (2018) Targeting integrin-linked kinase to suppress oncogenic KRAS signaling in Data availability Data generated during and/or analysed during the pancreatic cancer. Small GTPases 9(6):452–456. https://doi. current study are available from the authors upon request. org/10.1080/21541248.2016.1251383 Cutler ML, Bassin RH, Zanoni L, Talbot N (1992) Isolation of Rsp- Compliance with ethical standards 1, a novel CDNA capable of suppressing v-ras transformation. 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