Ercc1 Deficiency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Specific Manner
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Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Oncogenes and Tumor Suppressors Molecular Cancer Research Ercc1 Deﬁciency Promotes Tumorigenesis and Increases Cisplatin Sensitivity in a Tp53 Context-Speciﬁc Manner Mladen Jokic 1,2, Ignacija Vlasic 1,2, Miriam Rinneburger1,2, Niklas Klu € mper3, 4 3 3 Judith Spiro , Wenzel Vogel , Anne Offermann , Christiane Ku € mpers3, Christian Fritz , Anna Schmitt , Arina Riabinska , Maike Wittersheim5, 1,2 1,2 1,2 Sebastian Michels6, Luka Ozretic 5, Alexandra Florin5, Daniela Welcker1,2,7, Mehmet Deniz Akyuz , Michael Nowak9, Martin Erkel10, Ju 8 € rgen Wolf6, Reinhard Bu€ ttner , Bjo 5 € rn Schumacher , Ju 8 € rgen Thomale , Thorsten Persigehl4, 10 David Maintz , Sven Perner , and Hans Christian Reinhardt1,2 4 3 Abstract KRAS-mutant lung adenocarcinoma is among the most com- tumors that relapsed after cisplatin treatment in our model devel- mon cancer entities and, in advanced stages, typically displays op a robust etoposide sensitivity that is independent of the Ercc1 poor prognosis due to acquired resistance against chemotherapy, status and depends solely on previous cisplatin exposure. Our which is still largely based on cisplatin-containing combination results provide a solid rationale for further investigation of the regimens. Mechanisms of cisplatin resistance have been extensive- possibility of preselection of lung adenocarcinoma patients ly investigated, and ERCC1 has emerged as a key player due to its according to the functional ERCC1- and mutational TP53 status, central role in the repair of cisplatin-induced DNA lesions. How- where functionally ERCC1-incompetent patients might beneﬁt ever, clinical data have not unequivocally conﬁrmed ERCC1 status from sequential cisplatin and etoposide chemotherapy. as a predictor of the response to cisplatin treatment. Therefore, we employed an autochthonous mouse model of Kras-driven lung Implications: This study provides a solid rationale for the adenocarcinoma resembling human lung adenocarcinoma to stratiﬁcation of lung adenocarcinoma patients according to investigate the role of Ercc1 in the response to cisplatin treatment. the functional ERCC1- and mutational TP53 status, where Our data show that Ercc1 deﬁciency in Tp53-deﬁcient murine lung functionally ERCC1-incompetent patients could beneﬁt from adenocarcinoma induces a more aggressive tumor phenotype that sequential cisplatin and etoposide chemotherapy. Mol Cancer Res; displays enhanced sensitivity to cisplatin treatment. Furthermore, 14(11); 1110–23. 2016 AACR. 1 Department I of Internal Medicine, University Hospital of Cologne, Introduction Weyertal 115B, 50931, Cologne, Germany. 2Cologne Excellence Cluster on Cellular Stress Response in Aging-Associated Diseases, University Nucleotide excision repair (NER) is a complex DNA repair of Cologne, Weyertal 115B, 50931, Cologne, Germany. 3Pathology of system that has evolved to cope with helix-distorting lesions, the University Medical Center Schleswig-Holstein, Campus Luebeck and the Research Center Borstel, Leibniz Center for Medicine and such as those inﬂicted by nitrosamines, benzo(a)pyrenes Biosciences, 23538 Luebeck and 23845 Borstel, Germany. 4Depart- (from cigarette smoke), cyclobutane pyrimide dimers, and ment of Radiology, University Hospital of Cologne, Kerpener Str. 62, pyrimidine (6-4) pyrimidone photoproducts (induced by UV 50937, Cologne, Germany. 5Institute of Pathology, University Hospital of Cologne, Kerpener Str. 62, 50937, Cologne, Germany. 6Department I light), as well as crosslinks between guanine bases (induced of Internal Medicine, University Hospital of Cologne, Kerpener Str. 62, by the chemotherapeutic agents cisplatin, carboplatin, and 50937, Cologne, Germany. 7Department II of Internal Medicine, Uni- oxaliplatin; refs. 1–4). The NER mechanism consists of four versity Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. distinct steps, namely (i) recognition of the DNA lesion; (ii) 8 Institute for genome stability in ageing and disease, CECAD Research Center, Joseph-Stelzmann-Str. 26, 50931, Cologne, Germany. 9Insti- DNA unwinding, (iii) endonuclease-mediated incisions 30 tute of Pathology, University Hospital Bonn, Sigmund-Freud-Str. 25, and 50 of the lesion; and (iv) DNA resynthesis and ligation 53127, Bonn, Germany. 10Institute for Cell Biology, University Hospital (1–4). Lesion recognition occurs through one of two mechan- Essen, Hufelandstraße 55, 45122, Essen, Germany. isms, either global genome repair (GG-NER), which constant- Note: Supplementary data for this article are available at Molecular Cancer ly scans noncoding and nontranscribed regions of the Research Online (http://mcr.aacrjournals.org/). genome, or transcription-coupled repair (TC-NER), which M. Jokic, I. Vlasic and M. Rinneburger contributed equally to this article as ﬁrst is activated upon detection of stalled RNA polymerase II authors. (1–6). The ERCC1/XPF endonuclease complex constitutes a Corresponding Author: Mladen Jokic, Department I of Internal Medicine, critical component of the NER mechanism, which mediates University Hospital of Cologne, Weyertal 115B, Cologne 50931, Germany. the DNA incision 50 of the lesion (7, 8). In addition to its Phone: 4922-1478-96702; Fax: 4922-1478-96719; E-mail: contribution to NER, excision repair cross-complementation firstname.lastname@example.org group 1 (ERCC1) is involved in recombination-mediated doi: 10.1158/1541-7786.MCR-16-0094 DNA repair, as well as the repair of interstrand crosslinks 2016 American Association for Cancer Research. (9, 10). In humans, four distinct ERCC1 splice variants exist, 1110 Mol Cancer Res; 14(11) November 2016 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin of which only one (ERCC1-202) encodes a protein product adenocarcinomas (22). In addition, we combined a conditional that is capable of mediating repair of cisplatin-induced DNA Ercc1 allele (Ercc1ﬂox) (23) with the K and KP models to obtain damage (11). KrasLSL.G12D/wt;Ercc1ﬂox/ﬂox (KE) and KrasLSL.G12D/wt;Tp53ﬂox/ﬂox; Given its prominent role in NER-mediated repair of cisplatin- Ercc1ﬂox/ﬂox (KPE) mice, respectively. Mice were kept on a mixed induced DNA lesions, ERCC1 expression levels have been eval- C57Bl6/FVB background. To induce lung tumors, 8-week-old uated as a potential biomarker to predict the response of cancer KP mice were anesthetized with ketamine (100 mg/kg) and patients to cisplatin-based chemotherapy regimens. Although xylazine (20 mg/kg) by intraperitoneal injection, prior to intra- numerous studies indicate that low levels of ERCC1 mRNA or tracheal application of a replication-deﬁcient adenovirus expres- protein expression predict enhanced overall survival in response sing Cre [Ad-CMV-Cre, 2.5 107 plaque-forming unit (PFU)], to platinum-based chemotherapy in human non–small cell lung as described previously (22). Five weeks after Ad-CMV-Cre cancer (NSCLC; refs. 12–14), several groups have reported con- inhalation, lungs of KP and KPE mice were scanned by mCT tradictory results (11, 15–18). Thus, there is conﬂicting data on imaging (Aloka, Latheta LCT-100) under ketamine/xylazine anes- the role of ERCC1 expression as a predictor for cisplatin sensitivity thesia (as described above) to conﬁrm tumor formation. K and in human lung adenocarcinomas. Of note, the method of ERCC1 KE mice were imaged 12 weeks after tumor induction in the detection varied between the studies, where some used RT-PCR same manner. After mCT-conﬁrmed tumor formation, mice of all (14, 15, 18) and others used IHC (11–13, 16, 17) to determine four genotypes (K, KE, KP, and KPE) were treated with 7.5 mg/kg ERCC1 status. Particularly different batches of the commonly cisplatin (Sigma-Aldrich, P4394) once a week for 3 weeks. One used ERCC1 antibody 8F1 were found to deliver non-reproduc- week following administration of the last dose, mice were reim- ible results (11, 13). Further uncertainty was spurred by the recent aged by mCT to assess tumor response. The percentage of tumor discovery that 8F1 does not only engage ERCC1, but also cross- versus normal lung volume before and after cisplatin treatment reacts with another otherwise unrelated protein due to a common was analyzed in each animal, and the results were conﬁrmed epitope (19). These conﬂicting results, together with the lack of a by a specialized radiologist, using the semiautomatic segmenta- reliable and clinically applicable method of ERCC1 detection, led tion software Imalytics (Philips). Tumor tissue volume (mm3) to considerable confusion in the ﬁeld and a certain hesitance to and lung volume (mm3) were measured by regions of interest pursue ERCC1 as a potential biomarker. using a 3D Hounsﬁeld unit threshold technique, and the per- Despite the development of novel, molecularly targeted ther- centage (%) of tumor and normal lung volumes was calculated. apeutics for the treatment of many different cancer entities, In addition, acute tumor response was assessed 24 hours fol- cisplatin remains a cornerstone in the frontline regimens of most lowing administration of a single dose of cisplatin (7.5 mg/kg), solid tumors, including NSCLC, which is the most common using immunohistochemical staining for cleaved caspase-3 and malignancy after non-melanocytic skin cancer, with deaths from Pt-(GpG) adducts. Second line of chemotherapy based on etopo- lung cancer exceeding those from any other malignancy (20). side (Sigma-Aldrich, E1383, 20 mg/kg, 2 consecutive days per Cisplatin-based combination regimens are particularly important week, for 2 weeks, i.p.) treatment was applied on a cohort of KP for the treatment of KRAS-mutant lung adenocarcinoma, which (n ¼ 7) and KPE (n ¼ 10) mice that had previously undergone represents the largest subentity of lung adenocarcinoma and cisplatin treatment as described above. Relapse of the tumors for which no targeted therapeutic approach is currently available resistant to cisplatin was veriﬁed by mCT. Mice were treated with (20, 21). It is thus interesting to identify genetically deﬁned etoposide and monitored for survival. Mice used for the syngeneic scenarios in which KRAS-mutant tumors respond to cisplatin. allograft experiment (n ¼ 5) were anesthetized (2.5% isoﬂurane) Here, we employed genetically engineered mouse models of and injected with 106KPE (n ¼ 1) control cells at the upper part of Kras-driven lung adenocarcinoma to address the role of Ercc1 in the back and with 106KPE post-cisplatin cells (n ¼ 5) in the NSCLC tumorigenesis and cisplatin response. Conditional dele- lower part of the back. After tumor formation was veriﬁed by tion of Ercc1 in Kras-driven NSCLC models with concomitant palpation and mCT, mice were subjected to etoposide treatment Tp53 deﬁciency leads to more aggressive tumors that result in (20 mg/kg, 3 consecutive days per week, for 2 weeks, i.p.), and signiﬁcantly reduced overall survival than in Ercc1-proﬁcient tumor progression was monitored by mCT reimaging one week disease. In addition, we show that Ercc1-deﬁcient tumors display after the last etoposide treatment. Tumor volume was assessed by a massively enhanced cisplatin response. Finally, we demonstrate OsiriX, free and open-source DICOM viewer (OsiriX v.5.8.2, that in vivo cisplatin treatment of mice bearing conditionally Pixmeo). All animal procedures were approved by the local Ercc1-deﬁcient lung adenocarcinomas leads to the selection of animal protection committee and the local authorities. rare tumor cells that retain one functional allele of Ercc1. These cells then constitute relapsing tumors, which display an increased Mouse lung adenocarcinoma grading system sensitivity to etoposide. Together, our data suggest that KRAS- Formalin-ﬁxed parafﬁn-embedded (FFPE) mice lung sam- and TP53-mutant NSCLC patients could be stratiﬁed on the basis ples were cut into 4-mm thick sections and mounted on slides. of functional ERCC1 expression levels, as low functional ERCC1 After staining with hematoxylin and eosin (H&E), the tumors expression might be associated with favorable cisplatin response were assessed by a pathologist to evaluate the tumor burden. and sensitivity toward etoposide in the second line. A tumor grading system with three tumor grades (1, 2, and 3) based on the relative amount of diffuse or nodular tumor growth was applied. Grade 1 ("diffuse") tumors were deﬁned Materials and Methods by a predominantly diffuse tumor architecture (>90% diffuse). Autochthonous and allograft murine lung adenocarcinoma Grade 3 ("nodular") tumors predominantly show a nodular model growth pattern (>90% nodular), and in between, grade 2 KrasLSL.G12D/wt (K) and KrasLSL.G12D/wt;Tp53ﬂox/ﬂox (KP) mice ("mixed") contains tumors with a mixed architecture (>10% were used as autochthonous models of KRAS-mutant lung diffuse and >10% nodular). www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1111 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. A 100 KrasLSL.G12D/wt (K) 100 KrasLSL.G12D/wt; Trp53fl/fl(KP) KrasLSL.G12D/wt; Ercc1fl/fl(KE) KrasLSL.G12D/wt; Trp53fl/fl; Ercc1fl/fl(KPE) P = 0.571 75 75 ***P < 0.0001 Survival (%) Survival (%) 32d 50 50 25 25 0 0 0 60 90 120 150 180 210 240 0 60 90 120 150 Time (days) Time (days) B C 100 12 w after Ad-Cre Overall lung volume (%) Early-stage tumors 90 Normal tissue 80 Tumor tissue 70 60 50 40 30 20 10 Late-stage tumors 0 K KE KP KPE (n = 7) (n = 3) (n = 3) (n = 9) K KE KP KPE 5,000 µm 1,000 µm 100 µm G1 G2 G3 D P = 0.7878 P = 0.4512 100 Ercc1-positive index (%) 90 80 K 70 60 50 40 30 20 10 0 KE 4 wKE 12 wKE 12 w K (n = 4) (n = 3) (n = 3) Ercc1-negative cells Ercc1-positive cells 2,000 µm 1,000 µm 100 µm P = 0.7854 *P = 0.0178 100 Ercc1-positive index (%) 90 80 KP 70 60 50 40 30 20 10 0 KPE 4 wKPE 12 w KPE 12 w KP (n = 9) (n = 9) (n = 3) Ercc1-negative cells Ercc1-positive cells 2,000 µm 1,000 µm 100 µm Anti-Ercc1, 12 weeks post Ad-Cre 1112 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin Immunohistochemical staining and quantiﬁcation of protein Luminata Crescendo Western HRP Substrate (Millipore, expression in murine FFPE tissue WBLUR0100) on a Bio-Rad ChemiDoc MP System. Immunohistochemical staining against cleaved caspase-3 (CC3) and Ercc1 was conducted using the Ventana Discovery Clonogenic survival assay XT automated staining system (Ventana Medical Systems) on A total of 2 103 cells per condition were seeded in 6-cm FFPE mice lung samples. In brief, slides were incubated at room plates and incubated for 24 hours. Cells were either treated with temperature with the primary antibody according to the man- mock or were exposed to 5 mmol/L cisplatin for 24 hours, and ufacturers' instructions (dilutions, clones, manufacturer): anti- then the medium (RPMI) was replaced. Cells were incubated CC3 rabbit polyclonal (1:100, #9661, Cell Signaling Technol- for 10 days with medium exchange, ﬁxed, and stained with ogy) and anti-Ercc1 rabbit monoclonal (1:100, EPR7277, 0.5% crystal violet (Sigma, HT90132). Abcam). Antibody dilution was conducted using a Ventana diluent. Signal detection was done using the UltraMap anti-Rb Immunostaining and measurement of Pt-(GpG) adducts in HRP Detection Kit (Ventana Medical Systems). Finally, slides DNA were counterstained with hematoxylin and bluing reagent, Plated cells were treated with cisplatin (20 mg/mL) for 4 hours dehydrated, and mounted. Immunohistochemical stainings and were subsequently maintained in drug-free media for up to 48 were analyzed independently by two experienced observers hours. At various time points, cell aliquots were harvested, washed (S. Perner and N. Kl€ umper). in PBS, resuspended, and placed onto microscopic slides (Super- Quantiﬁcation of staining intensity by CC3 and Ercc1 anti- frost Plus Gold Adhesion Slides, Thermo Scientiﬁc). Lungs of bodies was performed using a semiquantitative image analysis tumor-bearing mice were perfused 24 hours after cisplatin treat- program (Deﬁniens Inc.) as described earlier (24). In short, the ment (7.5 mg/kg) with Tissue-Tek OTC/PBS solution (1:1; slides were scanned and areas containing lung adenocarcinoma Sakura), resected, and placed in liquid N2. Frozen tissue sections within the lung specimens were marked manually to exclude (12 mm) were prepared on adhesion slides. Pt-(GpG) intrastrand normal and stromal areas. Afterward, the software analyzed the cross-links in the nuclear DNA of single cells were visualized and staining intensity of tumor cells in the selected regions, and the measured by an immunocytologic assay using the Pt-(GpG)– cells were deﬁned as positive through a predeﬁned intensity speciﬁc antibody "R-C18" essentially as described previously threshold. The "positive index" was calculated as the ratio of (25). Brieﬂy, cells were ﬁxed in methanol (20 C), denatured positive cells divided by all cells in the region of interest. by alkaline treatment (60% seventy mmol/L NaOH/140 mmol/L NaCl; 40% methanol; 5 minutes, 0 C), and digested successively with pepsin (100 mg/mL; 10 minutes; 37 C) and proteinase K Cell culture (200 mg/mL; 10 minutes; 37 C). After blocking (5% skim milk in Murine lung adenocarcinoma tumor cells were isolated and PBS), slides were immunostained with "R-C18" rat anti-Pt-(GpG) cultured in RPMI containing 10% FBS. Retained Ercc1ﬂ allele in antibody (20 ng/mL in PBS/BSA; 12 hours; 4 C) and with Cy3- KPE post-cisplatin cells was knocked out (recombined) by treat- labeled rabbit anti-(rat Ig) antibody (Dianova) for 1 hour at 37 C. ment with 2.5 107 PFU of Ad-CMV-Cre in three rounds. Nuclear DNA was counterstained with DAPI (1 mg/mL in PBS). DAPI- and Cy3-derived signals were integrated and measured PCR separately for individual cell nuclei using a microscope-coupled For genotyping purposes, Ercc1ﬂox and Ercc1wt alleles were digital image analysis system (Zeiss Axioplan; ACAS 6.0 Image detected using ERCC1_F (50 -TAC TTG CCA GGG CAT TTG Analysis System, Ahrens Electronics). Antibody-derived ﬂuores- TGT-30 ) and ERCC1_R (50 -GTA TTG AGT GTT TTG CCT GCA cence signals were normalized to the corresponding DNA content T-30 ) primers. A 350-bp band indicated the Ercc1ﬂox allele, while a of the same nucleus and expressed as arbitrary ﬂuorescence units 500-bp band indicated the presence of the Ercc1wt allele. Recom- (AFU). Values were calculated as means of >100 measured cells bined Ercc1ﬂox allele was detected with primers F25731 and per sample, and error bars represent 95% conﬁdence intervals. For F25732 as described before (23). lung sections, randomly selected areas (100 100 mm) of tumor cells were measured and calculated for AFU values. Western blot analysis Murine lung adenocarcinoma cells were collected, lysed Immunoﬂuorescence [Invitrogen NP40-based lysis buffer (FNN0021) with protease For the staining of KPE and KPE post-cisplatin cells for g-H2AX, inhibitors], and quantiﬁed using the BCA Protein Assay Kit we seeded 105 cells per 18 cm2 coverslip and incubated for (Pierce). Protein extracts (30 mg) were subjected to 10% SDS- 24 hours. Cells were either mock treated or were exposed to PAGE, immunoblotted for Ercc1 (Abcam, EPR7277, 1:1,000) and 10 mmol/L etoposide for 60 minutes, and then the medium b-actin (Sigma-Aldrich, A5316, 1:5,000), and analyzed using the (RPMI) was replaced. Cells were collected at 6, 24, and 96 hours Figure 1. Ercc1 deletion enhances more aggressive tumor phenotype in Tp53-deﬁcient lung adenocarcinoma. A, survival of chemo-na€ve KrasLSL.G12D/wt (K) and KrasLSL.G12D/wt;Ercc1ﬂ/ﬂ (KE; left) and KrasLSL.G12D/wt;Tp53ﬂ/ﬂ (KP) and KrasLSL.G12D/wt;Tp53ﬂ/ﬂ;Ercc1ﬂ/ﬂ (KPE) mice (right) inhaled with Ad-CMV-Cre. P values were calculated using log-rank test. B, representative H&E staining of murine lung adenocarcinoma from KPE mice at 4 weeks (early-stage tumors) and of KPE mice at 12 weeks after tumor induction (late-stage tumors). C, top, distribution of tumor versus normal lung tissue in K, KE, KP, and KPE mice at 12 weeks after tumor induction; bottom, distribution of murine lung adenocarcinoma grades (G1, G2, G3) in corresponding K, KE, KP, and KPE mice at 12 weeks after tumor induction. D, left, anti-Ercc1 immunostaining of lung sections of K, KE, KP, and KPE mice at 12 weeks after tumor induction (three magniﬁcations for each genotype; right top, quantiﬁcation of Ercc1-positive tumor cells in KE mice (n ¼ 4) at 4 weeks after tumor induction, KE mice (n ¼ 3) at 12 weeks after tumor induction, and of control K mice (n ¼ 3) at 12 weeks after tumor induction; right bottom, quantiﬁcation of Ercc1-positive tumor cells in KPE mice (n ¼ 9) at 4 weeks after tumor induction, KPE mice (n ¼ 9) at 12 weeks after tumor induction, and of control KP mice (n ¼ 3) at 12 weeks after tumor induction. P values were calculated by comparing percentages using two-tailed t test. , P < 0.05. www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1113 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. A KP KPE **P = 0.0015 ***P = 0.0001 100 Pre-cisplatin Volume of tumor/lung tissue (%) 80 60 40 Post-cisplatin 20 0 KP pre cis KP post cis KPE pre cis KPE post cis B ***P < 0.0001 100 Fraction of apoptotic cells (%) KP 80 60 40 KPE 20 0 5,000 mm 1,000 mm 100 mm KP KPE Anti-CC3, 24 h post-cisplatin C 100 KrasLSL.G12D/wt; Trp53fl/fl(KP) KrasLSL.G12D/wt; Trp53fl/fl(KP) cis Survival (%) P = 0.356 75 50 25 0 75 100 125 150 175 Time (days) 100 KrasLSL.G12D/wt; p53fl/fl; Ercc1fl/fl(KPE) KrasLSL.G12D/wt; p53fl/fl; Ercc1fl/fl(KPE) cis Survival (%) 75 ***P < 0.0001 30.5 d 50 25 0 50 75 100 125 150 Time (days) Figure 2. Ercc1 deﬁciency sensitizes Tp53-deﬁcient lung adenocarcinoma to cisplatin in vivo. A, left, mCT-based monitoring of tumor response in KP and KPE mice prior to and after cisplatin treatment; right, quantiﬁcation of tumor volume in KP and KPE mice prior to and after cisplatin treatment. P values were calculated using two-tailed t test. B, left, representative anti–cleaved caspase-3 immunostaining of lung sections of KP and KPE mice after single cisplatin treatment; right, quantiﬁcation of the fraction of apoptotic cells in KP and KPE mice after single-dose cisplatin treatment. C, survival comparison of KP chemo-na€ve and KP cisplatin-treated mice (top) and of KPE chemo-na€ve and KPE cisplatin-treated mice (bottom). P values were calculated using log-rank test. 1114 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin A B **P = 0.0080 KPE 4 w 100 Ercc1-positive tumor cells (%) 80 60 40 KPE 12 w 20 0 4 w KPE 12 w KPE KPE chemo-naïve chemo-naïve cisplatin (n = 9) (n = 9) (n = 10) KPE cis Ercc1-negative cells Ercc1-positive cells 2,000 mm 1,000 mm 100 mm Anti-Ercc1 Figure 3. Cisplatin treatment selects for Ercc1 expression in lung adenocarcinoma. A, top, representative anti-Ercc1 immunostaining of lung sections of KPE mice at 4 weeks after tumor induction shown in three magniﬁcations; middle, representative anti-Ercc1 immunostaining of lung sections of KPE mice at 12 weeks after tumor induction shown in three magniﬁcations; bottom, representative anti-Ercc1 immunostaining of lung sections of KPE mice after three cycles of cisplatin treatment shown in three magniﬁcations. B, quantiﬁcation of Ercc1-positive tumor cells in KPE mice (n ¼ 9) at 4 weeks after tumor induction, KPE mice (n ¼ 9) at 12 weeks after tumor induction, and of KPE mice (n ¼ 10) after cisplatin treatment. P value was calculated by comparing percentages using a two-tailed t test. after etoposide removal and ﬁxed in 4% PFA for 15 minutes on minutes at 20 C. After several washing steps with PBS and room temperature. Cells were washed in PBS, permeabilized in 0.25% Triton X-100 or PBS and 1% BSA, cells were incubated at cytoskeleton buffer (10 mmol/L PIPES pH 6.8, 100 mmol/L NaCl, 4 C overnight with Puriﬁed Rabbit Anti-Active Caspase-3 primary 300 mmol/L sucrose, 3 mmol/L MgCl2, 1 mmol/L EGTA pH 8.0, antibody solution (BD Pharmingen, 559565, 1:200). Cells were and 0.5% Triton X-100) for 10 minutes on ice and then counterstained with propidium iodide (PI), and after 15-minute incubated in cytoskeleton stripping buffer (10 mmol/L Tris-HCl incubation in the dark, cells were assayed on Beckman Coulter pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 2% Tween 20, and Gallios Flow Cytometer. Cell populations were analyzed using 0.4% sodium deoxycholate) for 10 minutes on ice. Cells were Kaluza software and 50,000 to 100,000 events were counted in blocked in PBS supplemented with 5% BSA, 2% NGS, and each assay. 0.01% Triton X-100 for 1 hour at room temperature and incu- bated with anti-g-H2AX (phospho S140) [3F2] primary antibody solution (Abcam, ab22551, 1:250) at 4 C overnight. Results Cell nuclei were counterstained with Prolong Gold Antifade Conditional Ercc1 deletion enhances tumorigenesis in Reagent with DAPI (Molecular Probes, Life Technologies). Tp53-deﬁcient lung adenocarcinomas Cells were imaged on Axiovert 200M inverted ﬂuorescence imag- To directly validate Ercc1 deﬁciency as a predictor for cisplatin ing microscope equipped with a CCD camera (Zeiss) and with sensitivity in vivo, we aimed to assess the effect of Ercc1 deletion AxioVision Rel. 4.8 software. A total of 400 to 500 cells per in a model of Kras-driven lung adenocarcinoma. For this purpose, condition on several independent positions equally distributed we employed an established genetically engineered mouse over the slides were evaluated for g-H2AX positivity by AxioVision model, in which expression of oncogenic KrasG12D is pre- Rel. 4.8 software. vented through the insertion of a LoxP-ﬂanked transcriptional and translational STOP cassette in the endogenous locus Quantiﬁcation of apoptosis by ﬂow cytometry (KrasLSL.G12D allele, referred to as K in the following; ref. 22). For quantiﬁcation of apoptosis, 106 cells were seeded in 10-cm Intratracheal administration of adenoviral Cre recombinase dishes 24 hours prior to treatment. Cells were treated for 24 hours (Ad-CMV-Cre) leads to the expression of oncogenic KrasG12D with mock, 5 mmol/L cisplatin, 10 mmol/L etoposide, 0.1 mmol/L from its endogenous locus. In addition to this simple Kras- gemcitabine, or 10 mmol/L taxol. Next, cells were collected, driven model, we also used KrasLSL.G12D/wt;Tp53ﬂ/ﬂ mice (KP washed with ice-cold PBS, and ﬁxed in methanol for at least 30 in the following), in which both Tp53 alleles are ﬂanked by www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1115 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. WT MT REC A -500 bp 1 2 IoxP 3 4 5 IoxP 6 78 9 10 Ercc1fl -350 bp -250 bp b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 E WTc MTc Recc -500 bp 350 bp -250 bp b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 E WTc MTc Recc -500 bp 350 bp -250 bp c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 E WTc MTc Recc -500 bp 350 bp -250 bp c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 c11 c12 c13 c14 c15 E WTc MTc Recc -500 bp 350 bp -250 bp 1 2 3 4 5 6 7 8 Ercc1 (33 kDa) b-Actin (42 kDa) ***P < 0.0001 B C ***P < 0.0001 ***P < 0.0001 50 Fraction of apoptotic cells (%) 1.2 Pt-(GpG) in DNA (AFU) 1.0 40 KPE Cell lines 0.8 KP Cell lines 30 0.6 20 0.4 c6 10 0.2 0.0 0 0 12 24 36 48 KP KPE KPE KPE post cis post cis + Ad-Cre Time after cisplatin (h) D 3 + Ad-Cre 4 + Ad-Cre 5 + Ad-Cre 7 + Ad-Cre 8 + Ad-Cre 9 + Ad-Cre 1 2 3 4 5 1 6 7 8 9 Ercc1 (33 kDa) b-Actin (42 kDa) 1116 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin LoxP sites (22). Here, Ad-CMV-Cre drives expression of KrasG12D (59% 17.39 of Ercc1-positive tumor cells, n ¼ 3) after tumor and simultaneous deletion of both Tp53 alleles (22). The induction (Fig. 1D and Supplementary Fig. S1B). In stark impact of Ercc1 deletion was assessed by crossing conditional contrast, the Ercc1 deletion in Tp53-deﬁcient KPE animals was LoxP-ﬂanked Ercc1 alleles (23) homozygously into our K and almost complete [12.68 4.24% and 8.65 3.23% of Ercc1- KP models, yielding KE and KPE mice, respectively. The effect of positive tumors cells at 4 (n ¼ 9) and 12 weeks (n ¼ 9) after Ercc1 deletion was gauged through assessment of the overall tumor induction, respectively; Fig. 1D and Supplementary survival of K, KE, KP, and KPE animals that inhaled Ad-CMV- Fig. S1C]. These data suggest that Ercc1 deletion is selected Cre at 8 weeks of age. As shown in Fig. 1A, the survival of K (n ¼ against in p53-proﬁcient settings, while it is tolerated in the 11) and KE (n ¼ 22) animals was not statistically different. In absence of functional p53. Furthermore, concomitant Tp53 and contrast, KPE animals (n ¼ 23) displayed a signiﬁcantly Ercc1 deletion appears to promote the development of more reduced overall survival, compared with their Ercc1-proﬁcient aggressive lung adenocarcinomas (Fig. 1A). KP counterparts (n ¼ 15). To further dissect this observation, we next performed histologic tumor volume assessments at 4 Ercc1 deﬁciency increases cisplatin sensitivity in Tp53-deﬁcient and 12 weeks after Ad-CMV-Cre (Fig. 1B and C and Supple- lung adenocarcinomas in vivo mentary Fig. S1A). These experiments revealed that lungs of K The conﬂicting clinical data on the role of ERCC1 expression and KE animals were inﬁltrated with similar tumor volumes at as a predictive biomarker for cisplatin response and overall both 4 and 12 weeks following Ad-CMV-Cre inhalation (Sup- survival (11–18) have led to a hesitance in pursuing ERCC1 plementary Fig. S1A and S1C). Similarly, no difference between expression as a potential stratiﬁer for the selection of chemo- KP and KPE lungs was observed at 4 weeks (Supplementary Fig. therapeutic regimens in the treatment of NSCLC. To formally S1A). However, at 12 weeks, lungs derived from KPE animals address the potential impact of low-level ERCC1 expression in a displayed a substantially higher degree of tumor inﬁltration, relevant autochthonous mouse model of NSCLC in vivo, we compared with KP mice (73.34 13.92% vs. 26.66 next assessed the cisplatin response in tumor-bearing K, KE, KP, 7.64%; Fig. 1C). Consistent with the enhanced aggressiveness and KPE animals (Fig. 2 and Supplementary Fig. S2). CT-based of KPE tumors compared with KP tumors (Fig. 1A), we found imaging was used prior to initiation of treatment to conﬁrm the that KPE tumors were of substantially higher grade (G3) com- presence of neoplastic lesions formed 5 weeks following Ad- pared with their KP counterparts (Fig. 1C). No substantial CMV-Cre inhalation in KP and KPE mice and 12 weeks fol- difference was observed between K and KE tumors, although lowing Ad-CMV-Cre inhalation in K and KE mice (Fig. 2A and a trend toward a shift from grade 1 to 2 was detected in KE Supplementary Fig. S2A). Upon tumor formation, we admin- tumors. Both, K and KE tumors were entirely of low tumor istered three courses of cisplatin (7.5 mg/kg, i.p., once weekly). grade (G1 and G2; Fig. 1C). Therapeutic response was evaluated 7 days following the last Acute Ercc1 deletion leads to apoptotic cell death in murine cisplatin dose by mCT-based restaging. As shown in Supple- hepatocytes (26). Furthermore, p53 protein levels were found to mentary Fig. S2A, cisplatin treatment induced signiﬁcant tumor be elevated in Ercc1-deﬁcient murine liver-, kidney-, and brain regression in both K and KE animals (P ¼ 0.0051 and 0.0012, tissue (27). These observations suggest that Ercc1 deletion pro- respectively). KP lung adenocarcinomas were entirely resistant motes the induction of a p53 response. Given the proapoptotic and displayed signiﬁcant (P ¼ 0.0015) tumor growth following cellular outcome of p53 signaling (28), we hypothesized that three cycles of cisplatin exposure (Fig. 2A). This observation Ercc1 deletion is tolerable in a p53-defective background, while it is in agreement with previous experiments that indicated cis- is detrimental in a p53-proﬁcient context. platin resistance of the KP model (29). In stark contrast, KPE To directly assess Ercc1 deletion in tumors, we next per- tumors displayed a signiﬁcant (P ¼ 0.0001) volume reduction formed immunohistochemical staining of lungs isolated from at follow-up mCT. We note that this result reﬂects the average tumor-bearing KE and KPE animals 4 and 12 weeks following of multiple tumors, which may have discordant responses Ad-CMV-Cre inhalation with the adequate controls (K and with some tumors responding and others showing stable, or KP tumors 12 weeks after tumor induction, respectively). In even progressive disease. These CT-morphologic cisplatin KE tumors, we observed incomplete Ercc1 deletion at 4 (48.17 responses were mirrored by a highly signiﬁcant (P < 0.0001) 6.82% of Ercc1-positive tumor cells, n ¼ 4) and 12 weeks increase in cleaved caspase-3–positive apoptotic tumor cells in Figure 4. Ercc1 deﬁciency sensitizes Tp53-deﬁcient lung adenocarcinoma to cisplatin, in vitro. A, schematic representation of the Ercc1ﬂ allele showing primer positions for detection of the ﬂoxed allele (top gray arrows) and of the recombined allele [bottom black arrows; left; model partially taken from original publication (23)]. Schematic representation showing the positions of Ercc1 wild-type (WT), Ercc1ﬂ (MT), and Ercc1ﬂ recombined (REC) bands (right). Absence of Ercc1ﬂ allele in KPE tumor cells (b1-b14; top gel), presence of recombined Ercc1ﬂ allele in KPE tumor cells (b1-b14; second gel), presence of Ercc1ﬂ allele in KPE post-cisplatin (cis) cells (c1-c5 and c7-c15, except clone c6; third gel), and presence of recombined Ercc1ﬂ allele in KPE post-cisplatin cells (c1-c15; bottom gel). Anti-Ercc1 immunoblotting in KP cells (n ¼ 4, lanes 1–4) and KPE cells (n ¼ 4, lanes 5–8; top blot). Control b-actin immunoblotting in corresponding tumor cell lines (bottom blot). B, measurement of Pt-(GpG) adduct levels (AFU) in KP (n ¼ 2) and KPE tumor cell lines (n ¼ 3) at different time points after exposure to cisplatin. C, determination of the fraction of apoptotic cells (cleaved caspase-3 positive) using ﬂow cytometry in KP cell lines (n ¼ 19), KPE cell lines (n ¼ 14), KPE post-cisplatin cell lines (n ¼ 15), and KPE post-cisplatin pretreated with Ad-CMV-Cre (n ¼ 6), after cisplatin exposure in vitro. c6, clone 6. P values were calculated using a two-tailed t test. D, anti-Ercc1 immunoblotting in control KP cell line (lane 1), control KPE cell line (lane 2), KPE post-cisplatin, and corresponding KPE post-cisplatin cell lines treated with Ad-CMV-Cre (lanes 3 and 3 þ Ad-Cre, 4 and 4 þ Ad-Cre, 5 and 5 þ Ad-Cre; top left blot). Control b-actin immunoblotting in corresponding tumor cell lines (bottom left blot). Anti-Ercc1 immunoblotting in control KP cell line (lane 1), KPE post-cisplatin cell line clone 6 (lane 6), KPE post-cisplatin, and corresponding KPE post-cisplatin cell lines treated with Ad-CMV-Cre (lanes 7 and 7 þ Ad-Cre, 8 and 8 þ Ad-Cre, 9 and 9 þ Ad-Cre; top right blot). Control b-actin immunoblotting in corresponding tumor cell lines (bottom right blot). www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1117 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. KPE animals (n ¼ 4) compared with their KP counterparts (n ¼ zygous (Ercc1ﬂ/null) on the Ercc1 locus, as indicated by the presence 4; Fig. 2B). In line with this, exposure to cisplatin (single dose, of both Ercc1 ﬂoxed and Ercc1 recombined bands (c1–c5 and i.p., 7.5 mg/kg) leads to signiﬁcant increase of DNA Pt-(GpG) c7–c15). Only one of these 15 clones (clone c6), was homozy- adduct levels in KPE tumors (n ¼ 4) when compared with Ercc1- gously Ercc1-deﬁcient, as only the recombined allele was detect- proﬁcient KP tumors (n ¼ 3), in vivo (Supplementary Fig. S2B). able on the PCR. These observations were conﬁrmed by Ercc1 Furthermore, we monitored the survival of cisplatin-treated K immunoblotting (Fig. 4A and D). (n ¼ 11), KE (n ¼ 17), KP (n ¼ 23), and KPE (n ¼ 23) animals. We next asked whether the retained Ercc1 allele is necessary As shown in Supplementary Fig. S2C, median survival and sufﬁcient to mediate cisplatin resistance. For this purpose, of chemo-na€ve K and KE animals was 159 and 154 days, we initially compared the cisplatin response of cell lines iso- respectively. Cisplatin treatment substantially enhanced overall lated from KP tumors (KP cells) with those derived from survival by 51 and 76 days in the K and KE animals, respec- chemotherapy-na€ve KPE tumors (KPE cells). The ability of the tively. The cisplatin-induced survival gains did not signiﬁcantly different cell lines to clear cisplatin-induced DNA adducts was (P ¼ 0.165) differ between K and KE animals, consistent with assessed using a Pt-(GpG) intrastrand cross-link–speciﬁc mAb the incomplete Ercc1 deletion that we had observed histolog- that detects the most frequently occurring adduct formed by ically in the adenocarcinoma-bearing KE animals (Fig. 1D and cisplatin, which is associated with its cytotoxicity and antican- Supplementary Fig. S1B). A different picture emerged when we cer activity (25, 30). Exposure of both KP (n ¼ 5) and KPE analyzed the overall survival of animals bearing Tp53-deﬁcient (n ¼ 6) cell lines to cisplatin (20 mg/mL, 4 hours) led to the tumors. In agreement with previously published results, cis- occurrence of Pt-(GpG) adducts that remained present for up platin failed to signiﬁcantly (P ¼ 0.356) enhance the survival of to 48 hours after the initial insult in KPE cells (Supplemen- tumor-bearing KP animals, indicating that the KrasLSL-G12D/wt; tary Fig. S4A). To assess the kinetics of Pt-(GpG) adduct Tp53ﬂ/ﬂ-driven lung adenocarcinoma model mimics a chemo- removal, we next performed a time course experiment. As therapy-resistant high-risk clinical scenario (Fig. 2C; ref. 29). In shown in Fig. 4B, cisplatin treatment (20 mg/mL, 4 hours) in- marked contrast, cisplatin treatment signiﬁcantly prolonged duced the rapid occurrence of Pt-(GpG) adducts, in both KP the overall survival in KPE mice, compared with chemothera- (n ¼ 2) and KPE (n ¼ 3) cells, as early as 4 hours following drug py-na€ve (P < 0.0001) controls (Fig. 2C). Together, these data exposure. Ercc1-proﬁcient KP cells continuously cleared these indicate that Ercc1 deﬁciency signiﬁcantly enhances cisplatin adducts over time, and 48 hours following cisplatin treatment, sensitivity in aggressive Kras-driven and Tp53-deﬁcient lung adduct level was reduced by 52% in these cells. In marked adenocarcinomas, in vivo. However, cisplatin alone does not contrast, KPE cells completely failed to remove cisplatin appear to have curative potential in this model. Thus, we next adducts at the end of the 48-hour observation period (Fig. 4B). aimed to understand the mechanisms of acquired cisplatin We next asked whether KPE post-cisplatin cell lines isolated resistance in KPE animals. from KPE mice treated with cisplatin in vivo display cisplatin resistance, as one might predict based on their Ercc1 proﬁciency Ercc1 expression is selected in cisplatin-resistant lung (Fig. 4A and D). We exposed KPE post-cisplatin cells (n ¼ 15), adenocarcinomas as well as cells isolated from chemotherapy-na€ve KP (n ¼ 19) To assess the biological mechanisms of cisplatin resistance in and KPE (n ¼ 14) tumors to 5 mmol/L cisplatin for 24 hours. KPE animals, we performed histologic and immunohistochem- The fraction of apoptotic cells was assessed by ﬂow cytometry, ical analysis of KPE tumors that had relapsed after cisplatin using an antibody detecting cleaved caspase-3. As shown treatment (Fig. 3 and Supplementary Fig. S3). We speciﬁcally in Fig. 4C and Supplementary Fig. S4C, chemotherapy-na€ve stained tumor sections with an antibody detecting Ercc1, as we KPE cells were signiﬁcantly more sensitive to cisplatin than hypothesized that rare tumor cells that have undergone incom- their Ercc1-proﬁcient KP counterparts. In contrast, cells isolated plete recombination of the Ercc1ﬂ/ﬂ alleles might have escaped from KPE tumors that had relapsed after cisplatin treatment cisplatin treatment, similar to the KP tumors, which are entirely in vivo (KPE post-cisplatin cells) were almost entirely resistant resistant against cisplatin. Consistent with this hypothesis, tumors against cisplatin, with the exception of clone c6, which we that had relapsed after cisplatin exposure in KPE animals, stained had previously shown to be Ercc1 deﬁcient (Fig. 4A and D). strongly positive for Ercc1 (Fig. 3 and Supplementary Fig. S3). We Similar results were obtained when we assessed the long-term next aimed to verify that incomplete recombination of the Ercc1ﬂ/ﬂ effect of cisplatin treatment using clonogenic survival assays alleles in early tumors of KPE mice is the molecular explanation (Supplementary Fig. S4B). for Ercc1 expression in relapsed tumors. We further aimed to To directly test whether the retained Ercc1ﬂ allele in KPE post- validate that resistance in cisplatin-treated KPE tumors is indeed cisplatin cells that were isolated from cisplatin-treated KPE dependent on Ercc1 expression. For this purpose, we isolated tumors was responsible for the cisplatin resistance that we had cell lines from KP tumors (n ¼ 19; KP cells), as well as chemo- observed in vitro, we next deleted the retained Ercc1ﬂ allele, therapy-na€ve (n ¼ 14; KPE cells) and cisplatin-treated (n ¼ 15) using Ad-CMV-Cre, in vitro. Immunoblotting was used to verify tumors (KPE post-cisplatin cells) from KPE animals. To directly Cre-mediated deletion of Ercc1ﬂ (Fig. 4D). These cisplatin- assess recombination efﬁciency, we checked for the presence of experienced Ercc1null/null cells (KPE post-cisplatin þ Ad-Cre ﬂoxed and recombined Ercc1 alleles in chemotherapy-na€ve KPE cells) displayed a cisplatin-induced apoptotic response in vitro and in cisplatin-treated KPE post-cisplatin cell lines. As shown in that was indistinguishable from that observed in chemother- Fig. 4A, all 14 chemotherapy-na€ve KPE cell lines (b1–b14) apy-na€ve KPE cells (Fig. 4C). Altogether, these data strongly displayed complete Ercc1 recombination, indicated by the suggest that the retained Ercc1 allele in cell lines isolated from absence of the Ercc1 ﬂoxed (350 bp) and by the presence of KPE tumors that relapsed after cisplatin treatment was neces- Ercc1 recombined (250 bp) bands. In marked contrast, cisplatin- sary and sufﬁcient for the cisplatin resistance observed both treated KPE post-cisplatin cell lines were almost entirely hetero- in vitro and in vivo. 1118 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin Cisplatin-relapsed lung adenocarcinomas display increased this effect was even more apparent when the cells were expos- etoposide sensitivity ed to etoposide (10 mmol/L, 24 hours; Fig. 5A). To determine As NSCLC patients typically receive multiple lines of whether this etoposide sensitivity in the cisplatin-relapsed genotoxic chemotherapy, we next examined whether KPE KPE post-cisplatin cells is dependent on the Ercc1 status, we post-cisplatin cells that were isolated from tumors that had exposed KPE post-cisplatin cells pretreated with Ad-CMV-Cre relapsed after three cycles of cisplatin displayed differential in vitro (KPE post-cisplatin þ Ad-Cre; described in Fig. 4C) sensitivity to other chemotherapeutic agents. We speciﬁcally to etoposide (10 mmol/L, 24 hours; Fig. 5A, far right). We evaluated the cytotoxic effects of the spindle poison taxol, the observed no difference in the fraction of apoptotic cells nucleoside analogue gemcitabine, and the topoisomerase-II when compared with their Ercc1-proﬁcient counterparts (KPE inhibitor etoposide using ﬂow cytometry–based quantiﬁca- post-cisplatin cells), indicating that the observed sensitivity tion of apoptosis. As shown in Fig. 5, chemotherapy-na€ve and to etoposide is Ercc1 independent and therefore exclusively cisplatin-experienced KP and KPE cell lines did not display a cisplatin induced. differential taxol (10 mmol/L, 24 hours) sensitivity. In con- To further assess the differential cytotoxicity inﬂicted by trast, cisplatin-experienced KPE post-cisplatin cell lines show- etoposide on chemotherapy-na€ve and cisplatin-experienced ed sensitivity to gemcitabine (0,1 mmol/L, 24 hours), and KPE cells, we next quantiﬁed etoposide-induced genotoxic A Taxol, 10 mmol/L, 24 h Gemcitabine, 0.1 mmol/L, 24 h Etoposide, 10 mmol/L, 24 h Fraction of apoptotic cells (%) Fraction of apoptotic cells (%) Fraction of apoptotic cells (%) 50 50 50 40 40 40 30 30 30 20 20 20 10 10 10 0 0 0 KP KP KPE KPE KP KP KPE KPE KP KP KPE KPE KPE post cis post cis post cis post cis post cis post cis post cis + Ad-Cre DAPI g-H2AX DAPI gH2AX B Mock ***P < 0.0001 ***P < 0.0001 Fraction of gH2AX-positive cells (%) 100 80 6h 60 40 20 24 h 0 Mock 6h 24 h 96 h KPE cells KPE post cis cells 96 h KPE cells KPE post cis cells Figure 5. Cisplatin-relapsed tumor cells display etoposide sensitivity, in vitro. A, determination of fraction of apoptotic cells (cleaved caspase-3 positive) using ﬂow cytometry in KP, KP post-cisplatin (cis), KPE, and KPE post-cisplatin cell lines after exposure to taxol (left plot) and gemcitabine (middle plot). Determination of fraction of apoptotic cells (cleaved caspase-3 positive) using ﬂow cytometry in KP, KP post-cisplatin, KPE, KPE post-cisplatin cell lines, and corresponding KPE post-cisplatin cell lines pretreated with Ad-CMV-Cre in vitro after exposure to etoposide (right plot). B, Immunoﬂuorescence staining for g-H2AX in chemo-na€ve KPE and KPE post cisplatin cells. Left, foci were evaluated in mock, 6, 24 and 96 hours after etoposide exposure. Right, quantiﬁcation of fraction of g-H2AX-positive cells. P value was calculated by comparing percentages using two-tailed t-test. www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1119 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. A C KPE post cis cells KPE cells 5.0 4.5 2.0 Tumor growth (fold change) 1.5 B KPE KPE post cis 1 1.0 Pre Eto 0.5 0.0 –0.5 Post Eto –1.0 KPE tumors KPE post cis tumors D Ad-Cre Cisplatin 3x Etoposide 2x 0 5 6 7 Weeks KP/KPE mCT mCT mCT Tumor Tumor Tumor presence reduction relapse E 100 100 75 75 Survival (%) Survival (%) 30.5 d 24 d 50 50 25 25 0 0 75 100 125 150 175 50 75 100 125 150 Time (days) Time (days) KrasLSL.G12D/wt; Trp53fl/fl(KP) KrasLSL.G12D/wt; p53fl/fl;Ercc1fl/fl(KPE) P = 0.356 ***P < 0.0001 KrasLSL.G12D/wt; Trp53fl/fl(KP) cis KrasLSL.G12D/wt; p53fl/fl;Erccfl/fl(KPE) cis P = 0.154 **P = 0.0026 KrasLSL.G12D/wt; Trp53fl/fl(KP) 1st cis/2ndeto KrasLSL.G12D/wt; p53fl/fl;Erccfl/fl(KPE) 1st cis/2ndeto Pre cis Pre eto Pre cis Pre eto KPE KP 1120 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Ercc1 Loss Sensitizes Lung Adenocarcinoma to Cisplatin lesions, using indirect immunoﬂuorescence. We speciﬁcally cisplatin treatment of Ercc1-deﬁcient Kras-driven lung adeno- treated cells with etoposide (10 mmol/L, 60 minutes). Zero, carcinomas induces a hard-wired etoposide sensitivity that can 6, 24, and 96 hours following removal of etoposide, cells were be therapeutically exploited upon relapse following ﬁrst-line ﬁxed and stained with antibody detecting g-H2AX. Both che- cisplatin-based chemotherapy. motherapy-na€ve KPE and cisplatin-experienced KPE cells stained strongly positive for g-H2AX after 6 hours of etoposide exposure (Fig. 5B). While chemotherapy-na€ve KPE cells were Discussion largely devoid of nuclear g-H2AX foci 24 and 96 hours follow- ERCC1 deﬁciency in humans is rarely recognized and has been ing etoposide exposure (levels similar to KPE mock), cisplatin- implicated in severe developmental and neurodegenerative dis- experienced KPE post-cisplatin cells remained g-H2AX positive orders emphasizing the importance of ERCC1 that extends even 96 hours after the initial etoposide-induced insult. These beyond NER (31). In line with this, constitutive Ercc1 knockout observations suggest that in vivo cisplatin exposure leads to a mice show growth retardation and die several weeks after birth preserved impairment of the capacity to repair etoposide- due to severe liver damage and hepatocyte polyploidy (27, 32). induced DNA double-strand breaks in cisplatin-relapsed KPE These ﬁndings would suggest the importance of Ercc1 in the post-cisplatin cells. development, at least in certain tissues. However, the role of To validate the enhanced etoposide sensitivity that we ERCC1 in the tumor initiation and progression has been poorly observed speciﬁcally in cisplatin-experienced KPE cells, we next investigated. By employing a murine model resembling KRAS- performed a series of in vivo experiments. First, we transplanted driven human lung adenocarcinoma, we aimed to investigate a control chemotherapy-na€ve KPE cell line (n ¼ 1) and cis- whether lung cells that have undergone oncogenic transformation platin-experienced KPE post-cisplatin cells (n ¼ 5 distinct can tolerate loss of Ercc1. Our experiments show that complete cell lines) subcutaneously into syngeneic KPE recipient animals Ercc1 loss is only tolerated, when coupled with Tp53 deﬁciency (n ¼ 5) that had not been exposed to Ad-CMV-Cre and allowed and that this combined loss induces a more aggressive tumor tumors to form (Fig. 6A). Tumor onset was monitored by phenotype, leading to reduced overall survival of the affected palpation, and tumor volumes were quantiﬁed using mCT, prior animals. This ﬁnding corresponds to earlier studies showing to initiation of etoposide treatment (20 mg/kg, 3 consecutive that the genotoxic lesions, which are otherwise repaired through days per week, for 2 weeks, i.p.). Tumors that formed from an Ercc1-dependent mechanism, accumulate in liver and kidneys chemotherapy-na€ve KPE cells were entirely resistant and con- of Ercc1-deﬁcient mice (33). These lesions trigger the activation tinued to grow under etoposide exposure (Fig. 6B and C and of p53, resulting in early cell-cycle arrest and cellular senes- Supplementary Fig. S5). In sharp contrast, tumors that formed cence (33). Therefore, our results suggest that Kras-mutant and from cisplatin-experienced KPE post-cisplatin cells were exqui- Ercc1-deﬁcient murine lung adenocarcinoma might escape sitely sensitive to etoposide and shrunk under therapy (Fig. 6B these tumor-suppressive mechanisms in the absence of Tp53. and C and Supplementary Fig. S5). These allograft tumors thus Furthermore, the DNA repair deﬁciency associated with Ercc1 fully recapitulated our in vitro observations. loss might lead to a mutator phenotype that promotes the We next aimed to validate the enhanced etoposide sensitivity acquisition of additional mutations that ultimately cumulate in of KPE tumors that had relapsed following cisplatin treatment a more aggressive phenotype of the resulting tumors. in an autochthonous mouse model. For this purpose, we Acquired resistance to platinum-based chemotherapy is fre- generated KP (n ¼ 7) and KPE (n ¼ 10) animals, induced quently observed in human NSCLC, and only a small fraction lung adenocarcinomas through Ad-CMV-Cre inhalation, and of patients that were initially diagnosed with advanced administered three cycles of cisplatin (7.5 mg/kg, once a week stage disease survive 5 years beyond diagnosis (34). The for 3 weeks, i.p.), once mCT-conﬁrmed tumors had formed implication of the role of ERCC1 expression as a predictor (Fig. 6D and E). Upon mCT-conﬁrmed relapse, animals for a cisplatin sensitivity in human lung adenocarcinoma is received two courses of etoposide (20 mg/kg, 2 consecutive still not clear due to conﬂicting clinical data, and thus far to our days per week, for 2 weeks, i.p.). While this etoposide exposure knowledge, no in vivo lung adenocarcinoma study has did not lead to a signiﬁcant gain in overall survival in KP addressed this problem thoroughly. Moreover, human lung animals (P ¼ 0.154), KPE mice derived a substantial median adenocarcinomas harboring inactivating TP53 mutations are survival gain (24 days) and displayed a signiﬁcantly (P ¼ frequent (35) and are present in approximately half of the 0.0026) increased overall survival in response to sequen- KRAS-mutated lung adenocarcinoma tumors (36, 19). How- tial cisplatin/etoposide treatment, compared with cisplatin ever, their correlation with resistance to platinum-based che- treatment alone (Fig. 6E). Together, these data indicate that motherapy was not always straightforward (37–42). Here, we Figure 6. Cisplatin-relapsed tumors are sensitive to etoposide (Eto) in allograft and in autochthonous model. A, schematic representation of allograft experiment. Control KPE cell line (n ¼ 1) was injected into the upper back of each mouse (n ¼ 5). KPE post-cisplatin (cis) cell line (n ¼ 5) was injected into the lower back of a mouse (n ¼ 5). B, mCT-based monitoring of KPE and KPE post-cisplatin tumor response in allograft mice. Representation of resistant KPE tumor and sensitive KPE post-cisplatin tumor after exposure to etoposide (mouse 1 shown). C, top, quantiﬁcation of response of KPE and KPE post-cisplatin tumors to etoposide based on mCT imaging; bottom, macroscopic images of corresponding tumors (KPE tumors, left; KPE post-cisplatin tumors, right). D, schematic representation of autochthonous model treatment schedule. E, left, survival comparison of chemo-na€ve KP mice (black line), cisplatin-only treated KP mice (red line), and KP mice treated with cisplatin and after relapse with etoposide (purple line); right, survival comparison of chemo-na€ve KPE mice (black line), cisplatin-only treated KPE mice (red line), and KPE mice treated with cisplatin and after relapse with etoposide (purple line); bottom, mCT-based conﬁrmation of tumor presence before cisplatin and before etoposide treatment in KP mice (bottom left) and in KPE mice (bottom right). P values were calculated using log-rank test. www.aacrjournals.org Mol Cancer Res; 14(11) November 2016 1121 Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
Published OnlineFirst August 11, 2016; DOI: 10.1158/1541-7786.MCR-16-0094 Jokic et al. showed that cisplatin treatment speciﬁcally sensitizes aggres- tion therapy following tumor resection, there was no signiﬁcant sive Ercc1-deﬁcient Kras-mutant and p53-deﬁcient adenocarci- difference in overall survival, depending on the combination noma, while their Ercc1-proﬁcient counterparts remain resis- partner (etoposide, vinblastine, and vinorelbine; ref. 20). These tant, indicating that Ercc1 is a potent predictor of the outcome data may suggest that the effects that we observed may indeed to the cisplatin-based chemotherapy in lung adenocarcinoma, be selected by prior cisplatin treatment. Future experiments in vivo. These ﬁndings were further supported in vitro where we should address this question. showed that cisplatin-relapsed tumor cells expressed Ercc1 Altogether, our murine model faithfully resembling human through retained Ercc1 allele. In vitro deletion of this allele lung adenocarcinoma with differential ERCC1 expression pro- led to Ercc1 loss and resensitization against cisplatin. Our vides the basis for the investigation of the role of ERCC1 in the results are in line with studies performed on human tumor prediction to cisplatin sensitivity. We conclude that acquired cell lines, where cisplatin sensitivity was associated with low cisplatin resistance commonly observed in lung adenocarcino- total ERCC1 expression (43, 44), although the correlation to ma patients might be challenged by employing etoposide as a TP53 status is unclear. second line of chemotherapy to patients with low functional KRAS-mutant lung adenocarcinomas, which account for ERCC1 and TP53-mutated lung adenocarcinoma once cisplat- approximately 20% of all lung adenocarcinomas, are difﬁcult in-based chemotherapy fails. to treat, and targeted therapy has been proven to be clinically challenging (45). Chemotherapy regimens for metastasized Disclosure of Potential Conﬂicts of Interest NSCLC are largely based on cisplatin, usually applied in the No potential conﬂicts of interest were disclosed. combination with another compound. However, chemothera- py resistance almost inevitably occurs in these patients. Here, we aimed to explore the possibility of sequential instead of Authors' Contributions combined chemotherapy and therefore target relapsed murine Conception and design: M. Jokic, I. Vlasic, M. Rinneburger, J. Wolf, lung adenocarcinomas that emerge after cisplatin treatment. B. Schumacher, H.C. Reinhardt Development of methodology: M. Jokic, I. Vlasic, M. Rinneburger, First, we exposed relapsed tumor cell lines to three mechanis- N. Kl€umper, A. Riabinska, D. Welcker, M. Nowak, J. Thomale, T. Persigehl, tically different DNA-damaging agents in vitro, and we observed D. Maintz, S. Perner that these tumor cells show pronounced sensitivity to etopo- Acquisition of data (provided animals, acquired and managed patients, side by a mechanism not dependent on the Ercc1 status. This provided facilities, etc.): M. Jokic, I. Vlasic, M. Rinneburger, N. Kl€ umper, sensitivity was further corroborated by their inability to efﬁ- J. Spiro, C. Fritz, A. Schmitt, A. Riabinska, S. Michels, M.D. Akyuz, M. Nowak, ciently clear etoposide-induced double-strand breaks. Etopo- M. Erkel, J. Wolf, R. B€ uttner, J. Thomale, T. Persigehl, S. Perner Analysis and interpretation of data (e.g., statistical analysis, biostatistics, side is occasionally used for the treatment of NSCLC as an computational analysis): M. Jokic, I. Vlasic, M. Rinneburger, J. Spiro, additive drug to cisplatin (46), but not as a main drug choice A. Offermann, C. K€ umpers, M. Wittersheim, L. Ozretic, M. Erkel, J. Wolf, once the cisplatin chemotherapy fails. Therefore, we next aimed R. B€uttner, J. Thomale, T. Persigehl, S. Perner, H.C. Reinhardt to validate our observations in vivo by creating an in vivo Writing, review, and/or revision of the manuscript: M. Jokic, I. Vlasic, syngeneic model, in which we showed sensitization of trans- M. Rinneburger, N. Kl€ umper, J. Spiro, M. Wittersheim, J. Wolf, R. B€ uttner, planted cisplatin-relapsed cells to etoposide exposure. In line T. Persigehl, D. Maintz, S. Perner, H.C. Reinhardt Administrative, technical, or material support (i.e., reporting or organiz- with this, we were able to target cisplatin-relapsed tumors in an ing data, constructing databases): M. Rinneburger, A. Florin, T. Persigehl, autochthonous in vivo lung adenocarcinoma model where the D. Maintz, S. Perner, H.C. Reinhardt survival of KPE mice was signiﬁcantly prolonged after etopo- Study supervision: B. Schumacher, H.C. Reinhardt side treatment as a second line of therapy when compared with Other (IHC, HE staining, etc.): W. Vogel KPE mice exposed only to a single line of cisplatin chemother- apy. Conversely, KP mice that developed initially cisplatin- Grant Support resistant tumors did not experience tumor sensitivity to sub- This work was supported by the Volkswagenstiftung (Lichtenberg Pro- sequent double-strand break insult. These ﬁndings may indi- gramA107062, to H.C. Reinhardt), the Deutsche Forschungsgemeinschaft cate a requirement of initial Ercc1-dependent cisplatin sensi- (RE 2246/7-1, RE 2246/2-1 to H.C. Reinhardt), the Bundesministerium f€ ur tivity for the occurrence of subsequent sensitivity to etoposide Bildung und Forschung (BMBF 01ZX1303A, to H.C. Reinhardt), the Boeh- that is Ercc1 independent. However, we note that we did not ringer-Ingelheim Stiftung (Exploration Grants-Program, to H.C. Reinhardt), the Helmholtz-Gemeinschaft (PCCC, to H.C. Reinhardt), the Else Kr€ oner- directly examine the effect of combined cisplatin/etoposide Fresenius Stiftung (EKFS-2014-A06, to H.C. Reinhardt), and the Deutsche treatment in our models. Thus, we cannot formally exclude Krebshilfe (222521, to H.C. Reinhardt). the possibility that a sequential approach is preferable to The costs of publication of this article were defrayed in part by the payment of concurrent cisplatin/etoposide regimens. In fact, the interaction page charges. This article must therefore be hereby marked advertisement in between cisplatin-induced DNA damage and etoposide sensi- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tivity may also be immediately synergistic rather than a trait that is selected upon cisplatin exposure. However, at least in Received March 18, 2016; revised June 18, 2016; accepted July 6, 2016; NSCLC patients receiving adjuvant cisplatin-based combina- published OnlineFirst August 11, 2016. References 1. Bowden NA. Nucleotide excision repair: why is it not used to predict 3. Hoeijmakers JH. DNA damage, aging, and cancer. N Engl J Med 2009; response to platinum-based chemotherapy? Cancer Lett 2014;346:163–71. 361:1475–85. 2. Hoeijmakers JH. Genome maintenance mechanisms for preventing cancer. 4. Friedberg EC. How nucleotide excision repair protects against cancer. Nature 2001;411:366–74. Nat Rev Cancer 2001;1:22–33. 1122 Mol Cancer Res; 14(11) November 2016 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on February 23, 2021. © 2016 American Association for Cancer Research.
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