Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1

Oncotarget67033 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 41 Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1) Marco Perez1, Javier Peinado-Serrano1, Jose Manuel Garcia-Heredia1,2, Irene Felipe-Abrio1, Cristina Tous1, Irene Ferrer1, Javier Martin-Broto1,3, Carmen Saez1,4, Amancio Carnero1 1Instituto de Biomedicina de Sevilla, IBIS, Hospital Universitario Virgen del Rocio, Universidad de Sevilla, Consejo Superior de Investigaciones Cientificas, Seville, Spain 2Department of Vegetal Biochemistry and Molecular Biology, University of Seville, Seville, Spain 3Department of Medical Oncology, Virgen del Rocío University Hospital, Seville, Spain 4Department of Pathology, Virgen del Rocío University Hospital, Seville, Spain Correspondence to: Amancio Carnero, email: [email protected] Keywords: MAP17, bortezomib, PDX, sarcomas, biomarker Received: March 08, 2016 Accepted: August 09, 2016 Published: August 22, 2016 ABSTRACT Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness, and new treatment approaches are needed. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematological malignancies and in breast tumors. Therefore, we analyzed the expression of this oncogene in sarcomas and its relationship with clinico-pathological features, as well as tested whether it can be used as a new biomarker to predict the therapeutic response to bortezomib and new therapies for sarcomas. We found that the levels of MAP17 were related to clinical features and poor survival in a cohort of 69 patients with different sarcoma types, not being restricted to any special subtype of tumor. MAP17 expression is associated with poor overall survival (p<0.001) and worse disease-free survival (p=0.002). Cell lines with high levels of MAP17 show a better response to bortezomib in vitro. Furthermore, patient-derived xenografts (PDX) with high levels of MAP17 respond to bortezomib in vivo. Our results showed that this response is due to the lower levels of NFκB and autophagy activation. Therefore, we suggest that MAP17 is a new biomarker to predict the efficacy of bortezomib as a new therapy for sarcomas. INTRODUCTION Sarcomas are rare tumors of mesenchymal origin [1, 2, 3]with high cancer mortality in children and young adults. The vast majority of sarcomas are sporadic, with the existence of a few genetically linked cancer syndromes. Furthermore, some types of environmental exposure have been associated with specific types of sarcoma [2, 3]. Taxonomical analysis of sarcomas has identified approximately 60 subtypes of sarcoma, as well as more than 50 benign tumor subtypes [4, 5]. Sarcomas are usually grouped into two broad categories according to molecular genetics: sarcomas harboring a diploid or nearly diploid karyotype and simple genetic driver alterations, such as Ewing’s sarcoma, or sarcomas with a complex and imbalanced karyotype, such as osteosarcoma. The subgroups include very different clinical entities and are broadly drawn, not reflecting the genetic diversity among tumors of a given type or subtype or their diverse tumor biology [4]. Surgery is the standard approved treatment for most resectable sarcomas. Radiotherapy is selected Research Paper Oncotarget67034 www.impactjournals.com/oncotarget for those unresectable or residual tumors after surgery. Chemotherapy is used in metastatic disease [6]. However, many tumors exhibit chemoresistance, and new treatments are needed. Systemic therapies for sarcomas include doxorubicin, gemcitabine, ifosfamide and the recently accepted drug trabectedin [7–10]. To date, few direct targets for therapy have been demonstrated in sarcomas, in contrast to those for epithelial cancers. The exceptions are GISTs, in which imatinib, the c-Kit kinase inhibitor, induces a partial response or stable disease in most sarcoma patients [4]. These findings support the hypothesis that widely diverse sarcoma tumors may share a dependence on a particular protein and that this marker may, therefore, be expected to be effective for all histological subtypes that are positive for this biomarker. We have recently extended this observation for CDK inhibition. We have found that sarcoma tumors with combined high levels of CDK4 mRNA and low levels of p16INK4a, respond to palbociclib (a CDK4 inhibitor) in a panel of PDX models in vivo, irrespective of the tumor type [11]. MAP17 (DD96, PDZKIP1), a membrane-associated protein with a molecular weight of 17 kD [12–14], is expressed only in the cells of the proximal tubule of the adult kidney [15–17]. It contains two transmembrane regions and a hydrophobic N-terminus encoding one PDZ-binding domain [18, 19]. Through this domain, MAP17 has been demonstrated to bind proteins containing PDZ domains, including PDZK1 (NHRF3), and other NHRF-related proteins, such as NaPiIIa and NHe3 [15, 17, 19]. MAP17 increases the cellular reactive oxygen species (ROS), which have been shown to enhance the malignant properties of tumor cells [20–22]. Tumor cells overexpressing MAP17 show increased proliferation, reduced apoptosis, increased formation in soft agar and increased growth of tumors in nude mice [20, 22]. However, the inhibition of ROS by antioxidant treatments can prevent the enhancement of the pro-tumorigenic properties of cells [14]. MAP17 is overexpressed in most human carcinomas and in other non-epithelial neoplasias such as glioblastomas or lymphomas [23]; additionally, its expression is associated with progression [17, 23, 24]. While adenomas and benign tumors, as well as normal tissues, rarely express MAP17, a high proportion (50 to 90%) of late-stage or metastatic tumors show high levels of MAP17, correlating with a more de-differentiated phenotype [17, 22, 24–26]. These findings highlight the relevance of this gene in the process of tumorigenesis and development of tumors. MAP17, through an as-yet-unknown mechanism, increases ROS in cells. This ROS increase may be responsible, at least partly, for the enhanced malignant properties induced in tumor cells [33]. Because ROS is a potent proapoptotic insult, a further increase of ROS might switch the balance towards apoptosis. Thus, tumors with high MAP17 levels may respond to therapies increasing ROS. Therefore, MAP17 expression levels may be a suitable biomarker for prognosis. Cervix tumor patients treated with cisplatin and radiotherapy showed better survival if the tumor showed high levels of MAP17 [24, 27, 28]. Therefore, MAP17 is not only a marker for stage and malignant status but also may be a marker of response to therapies, inducing oxidative stress. In breast tumor cells, the MAP17 expression level determines the sensitivity to bortezomib by inhibiting the cytoprotective effects resulting from bortezomib-induced NFκB nuclear translocation and autophagy [29]. Furthermore, the inhibition of oxidative stress abolishes the MAP17- induced sensitivity to bortezomib. Therefore, high levels of MAP17 could be used for predicting bortezomib treatment responses in patients. In this manuscript, we have explored the relevance of the presence of MAP17 in sarcoma tumors where the primary response is mainly achieved by treatments with radiotherapy. We found that, in contrast to laryngeal and cervix adenocarcinomas, the expression of MAP17 is a poor prognostic factor. Therefore, as we are looking for new therapeutic modalities for sarcomas, we have tested whether high levels of MAP17 may be a determinant in the response to bortezomib. RESULTS Clinical cohort description The patient cohort with a clinical follow up had equal distribution by gender with 53% males and 47% females, with an average of 51 years at the time of diagnosis (Table 1). However, they showed a broad distribution in age, from 20 to 72 years. The histology was very variable with liposarcoma at 18.8% (n=13), undifferentiated pleomorphic sarcoma at 17.4% (n= 12) and leiomyosarcoma at 31.9% (n=22) as the most prevalent. Other types of minor represented sarcomas in our cohort were hemangiopericytoma at 4.3% (n=3), synovial sarcoma at 4.3% (n=3), neurogenic sarcoma at 4.3% (n=3), fibrosarcoma at 7.2% (n= 5), fibromyxoid sarcoma at 1.5% (n=1), angiosarcoma at 1.5% (n=1), and mesenchymal sarcomaat1.5% (n=1). These tumors were mostly metastatic at the time of diagnosis (76%), and the remainder was locally advanced. The treatment was doxorubicin 75 mg/m2 3w x 6 cycles in all cases with half of the cases in combination with trabectedin 1.1 mg/m 2 + doxorubicin 60 mg/m2 3w x 6 cycles 49.3% (n=34). This cohort has been reported in Martin-Broto et al [30]. Only for IHC analysis of MAP17 expression was this series completed with complementary samples (for which we do not have clinical information) used to assess MAP17 values in different sarcoma types (Supplementary Table S1). Oncotarget67035 www.impactjournals.com/oncotarget Table 1: Clinical characteristics of the patients Patients (N=69) Gender Male 53.6 (37) Female 46.4 (32) Age (years) 51 [20-72] Histology Liposarcoma 18.8 (13) Undifferentiated Pleomorphic Sarcoma 17.4 (12) Haemangiopericytoma 4.3 (3) Leiomyosarcoma 31.9 (22) Synovial Sarcoma 4.3 (3) Neurogenic Sarcoma 4.3 (3) Fibrosarcoma 7.2 (5) Unclassified Sarcoma 5.8 (4) Fibromyxoid Sarcoma 1.5 (1) Angiosarcoma 1.5 (1) Condrosarcoma 1.5 (1) Other 1.5 (1) Staging Locally advanced 23.2 (16) Metastatic 76.8 (53) Treatment Doxorubicin 75 mg/m2 3w x 6 cycles 50.7 (35) Trabectedin 1.1 mg/m2 + Doxorubicin/ 3w 60 mg/m2 3w x 6 cycles 49.3 (34) MAP17 Expression in human sarcoma We analyzed 69 samples, for which we had clinical information, for MAP17 expression by immunohistochemistry (Figure 1), finding the percentage of tumors with high levels of expression as in other tumor types [17]. Using 0,75 as the cut-off level of MAP17 expression as depicted by the ROC curve (Figure 2), with a sensitivity = 0,76 and a specificity = 0,56, we found that high levels of MAP17 are predictive of a worse diseasefree survival (DSF, p=0,03) and worse progression-free survival (PSF, p=0,033); however, there was no relevance regarding overall survival (OS) (Figure 2). These patients were treated with doxorubicin alone or with doxorubicin plus trabectedin. The analysis of the DFS, PFS or OS in these patients sorted by treatment scheme shows a similar trend in patients treated only with doxorubicin (Supplementary Figure S1A); in patients treated with the combination of doxorubicin plus trabectedin, MAP17 did not show any prognostic relevance (Supplementary Figure S1B). One hundred one tumor samples were analyzed for MAP17 expression by immunohistochemistry. Of the 101 samples, 39% showed higher levels of MAP17 protein expression and were considered positive for MAP17 expression (Figure 3A) according to the previous ROC curve performed. The distribution of tumors with MAP17 was broad and was not restricted to any specific sarcoma type (Figure 3B), but there was a direct correlation of the levels of MAP17 with tumor grade (ANOVA, p<0.05) (Figure 3C), which was statistically significant. The surrounding normal tissue did not express MAP17 or expressed very low levels. The analysis of the impact on DFS, PFS or OS on specific sarcoma types such as liposarcoma, undifferentiated or leiomyosarcoma, showed a similar trend to the total population comprising all sarcoma types (Supplementary Figure S2). However, the small number of cases precludes any strong conclusion. Furthermore, the analysis of databases for the expression of MAP17 in other types not sufficiently represented in our cohort, such as osteosarcoma or Ewing’s sarcoma with a high Oncotarget67036 www.impactjournals.com/oncotarget Figure 2: Relationship of the MAP17 levels with the clinical response in sarcomas. ROC curve for MAP17 showing that, at the cut-off point of MAP17>0,75, the sensitivity=0.76 and thespecificity=0.56. Correlation of the MAP17expression was measured as a dichotomous variable: MAP17 high rates (>0,75) with overall survival (OS), disease-free survival (DFS) and progression free survival (PFS). Figure 1: Representative pictures of MAP17-stained sarcoma samples. Angiosarcoma (AS), Leiomyosarcoma (Ly), GIST (Gist), pleomorphic sarcoma (Plm), chondrosarcoma (CS), Ewing’s sarcoma (EW). Oncotarget67037 www.impactjournals.com/oncotarget MAP17 level, also shows a worse prognosis in either OS or metastasis-free survival (Supplementary Figure S3). Therefore, our data showed that 40% of sarcomas display high levels of MAP17 protein at the time of diagnosis, unrelated to the specific tumor type. High levels of MAP17 are related to a higher grade and are predictive of a poor prognosis in patients with sarcoma. Furthermore, multivariate analysis indicated that MAP17 is an independent predictor of the sarcoma outcome (Table 2). The response to bortezomib in sarcoma cell lines correlates with MAP17 Levels Because these patients with high MAP17 showed a worse prognosis, we wondered whether we can find an alternative treatment for these patients. To this end, we planned to test bortezomib, which showed efficacy in breast tumor cells if MAP17 is overexpressed [29]. Thus, we first analyzed the role of MAP17 levels in sarcoma cell lines. To explore the effect of MAP17 on the response to bortezomib, we used a panel of 16 sarcoma cell lines of heterogeneous origin [11, 31, 32]. We treated them at different concentrations of bortezomib and obtained an IC50 value for each one. All of the responses were in the nM range (Table 3), distributed however in a 50-fold range, between 10 and 500 nM. In parallel, we measured the basal levels of MAP17 mRNA in all cell lines (Table 3) and found different expression levels between them. We distributed the cell lines into three groups according to the MAP17 mRNA level: those with barely detectable MAP17 mRNA (BP, CE, CD0024, SAOS2, SK-UT-1, HT- 1080 and CP0038), those with low but clearly detectable levels of MAP17 mRNA (AA, AW, BG and BD) and those with high levels of expression (BC, AZ, AX, SW872 and 93T449). The analysis of the correlation of the MAP17 mRNA levels and IC50 to bortezomib showed a trend Figure 3: MAP17 expression in sarcoma tumors. A. Overall distribution of MAP17-positive sarcomas. B. MAP17 level distribution among different sarcoma types. Angiosarcoma (AS), fibromyxoid sarcoma (FM), fibrosarcoma (F), hemangiopericytoma (HP), leiomyosarcoma (Ly), liposarcoma (L), neurogenic sarcoma, MPNST (N), synovial sarcoma (Sy), undifferentiated pleomorphic sarcoma (UPl), Ewing’s sarcoma (Ew), rhabdomyosarcoma (R), fusocellular sarcoma (Fs), GIST (G), pleomorphic sarcoma (Plm), and chondrosarcoma (CS). C. Map17 levels correlate with tumor grade in sarcoma. The mean values are represented for grade 1 (=0,45), grade 2 (=0,66) and grade 3 (0,90). ANOVA was performed to establish the statistical association between the MAP17 protein levels and grade of the tumor (p<0,05). Oncotarget67038 www.impactjournals.com/oncotarget (ANOVA, p=0,1) indicating that cells with higher levels of MAP17 are more sensitive to bortezomib than cells without MAP17 (Figure 4A). To functionally confirm these data, we overexpressed MAP17 cDNA in 2 sarcoma cell lines with very low expression, AA and AW (Figure 4B), and calculated a new IC50 to bortezomib in these cell lines and compared to parental cells expressing empty vector only. We found that the ectopic expression of MAP17 increases the sensitivity to bortezomib 2-3 fold (Figure 4C). To fully confirm these results, we reduced the levels of MAP17 in the AX sarcoma cancer cell line, which shows high endogenous levels of MAP17. We overexpressed an shRNA against MAP17 and reduced the levels of MAP17 to 50% (Figure 4D), and then we tested the bortezomib sensitivity (Figure 4E). We observed a 50% reduction in Table 3: IC50 to bortezomib of the panel of sarcoma cell lines used in this study and its correlation to the levels of MAP17 mRNA measured by RT-QPCR Cell line Tumor of origin IC50 Bortezomib (μM) MAP17 (2-ΔCT) 93T449 Liposarcoma 35,9±3,75 0,000182199 AA Leiomyosarcoma 20,65±0,75 6,21882E-05 AW Liposarcoma 30,95±8,5 0,00011437 AX Liposarcoma 19,3±5,2 0,000534323 AZ Fibrosarcoma 92,89±0,92 0,001050201 BC MPNST 32,5±9,8 0,00025699 BD Ewing’s Sarcoma 149,7±17,42 0,000111012 BG Myxoid Fibrosarcoma 28,99±5,8 7,64036E-05 BP Osteosarcoma 502±56,6 2,32244E-05 CE Rhabdomyosarcoma 15,53±2,9 4,55244E-05 CP0024 Leiomyosarcoma 30,75±5,46 2,84148E-05 CP0038 Leiomyosarcoma 43,65±9,78 2,52213E-05 HT1080 Fibrosarcoma 15,88±2,37 1,68955E-05 Saos-2 Osteosarcoma 52,78±9,5 1,30192E-05 SKUT1 Uterine Leiomyosarcoma 10,95±2,46 4,58532E-05 SW872 Liposarcoma 10,13±2,36 0,000262208 IC50 is the average of 3 independent experiments performed in triplicate. To measure human MAP17 RNA expression, real-time PCR was performed using a ABI 7900HT (Applied Biosystems), and we used GADPH as internal control/ reference. Quantitative and statistical analyses of the QPCR data were calculated using Applied Biosystem RQ Manager 1.2.1 software. We presented the 2eΔΔCT data. The levels of MAP17 mRNA presented are the average of the mRNA levels obtained in three independent determinations performed in triplicate samples. Table 2: Multivariate analysis. MAP17 is an independent predictor of other clinical variables DFS PFS OS Pearson correlation p-value Pearson correlation p-value Pearson correlation p-value MAP17 -0.268 0.015 -0.268 0.016 -0.142 0.129 Radiotherapy -0.155 0.134 -0.143 0.154 0.162 0.123 Differentiation -0.216 0.06 -0.167 0.116 -0.245 0.038 Metastatic disease -0.191 0.085 -0.125 0.187 -0.207 0.069 T4 extension -0.114 0.208 -0.157 0.130 -0.207 0.069 Oncotarget67039 www.impactjournals.com/oncotarget the sensitivity, comparable to the 2-fold increase observed in the cell lines with enforced overexpression of MAP17. Sarcomas overexpressing MAP17 are more sensitive to bortezomib in vivo Therefore, we decided to test the response of sarcomas to bortezomibin patientderived xenografts, PDXs, in vivo. Of a panel of sarcoma PDXs [11], we selected 4 models (Figure 5A) with different levels of MAP17 (Figure 5B). Twelve mice were implanted for each PDX model and were randomly distributed for the treatment with bortezomib or solvent alone (PBS). We found that the PDX with high levels of MAP17 showed a better response to bortezomib (Figure 5C). The tumor disappeared from the flank of all mice treated with bortezomib, and the mice survived. The PDX model S11, with still significant but low levels of MAP17, partially responded to bortezomib by decreasing the tumor growth rate and increasing survival by 25% approximately (Figure 5C). However, the models with low or no MAP17 (S14 and S29) did not respond to bortezomib (Figure 5C). Thus, our data in vivo, in PDX models of sarcoma, support our data in vitro and suggest that bortezomib may be a suitable therapy for sarcoma patients with high MAP17 at diagnosis. We have previously shown that MAP17 prevents cytoprotective NFκB activation and autophagy induced by bortezomib in breast tumor cells [29]. Therefore, we have tested whether these molecular markers correlate also in our PDX models prior to treatment (Figure 6A). S16, the PDX model that responds to bortezomib, showed lower levels of endogenous active NFκB (measured as p65 phosphorylated at Ser536) and autophagy (measured as p62 and/or LC3 increase), thus confirming the mechanistic role of these factors in the bortezomib response. Similar data could be observed in the AA and AW cell lines, where the overexpression of MAP17 reduces NFκB activation Figure 4: Correlation of the MAP17 levels with the sensitivity to bortezomib. A. Correlation of the MAP17 levels with the sensitivity to bortezomib in a panel of 16 sarcoma cell lines. The cell lines were subdivided according to the levels of MAP17: no or barely detectable levels of MAP17 mRNA= Low (BP, CE, CP0024, SAOS2, SK-UT-1, HT-1080 and CP0038); cells with low but clearly detectable levels of MAP17 mRNA= medium (AA, AW, BG and BD); and high levels of expression= high (BC, AZ, AX, SW872 and 93T449). The analysis of the correlation of MAP17 mRNA levels and IC50 to bortezomib was performed with ANOVA, p=0,1. B. Levels of MAP17 mRNA in cell lines ectopically expressing MAP17 cDNA (MAP17) or vector only (V). The data show the average levels of MAP17 mRNA by Q-RT-PCR, performed in three independent determinations. C. Curves showing the IC50 of cell lines ectopically expressing MAP17 cDNA (MAP17) or vector only (V). IC50 in the inset is the average ± SD from 4 independent experiments performed in triplicate. D. Decrease of the levels of MAP17 mRNA by specific expression of shmRNS against MAP17 in the sarcoma cell line AX. The data show the average levels of MAP17 mRNA by Q-RT-PCR, performed in three independent determinations. E. Curves showing the IC50 of cell lines ectopically expressing MAP17 sh mRNA (shM17) or scramble shRNA as control (Scr M17). IC50 in the inset is the average ± SD from 3 independent experiments performed in triplicate. Oncotarget67040 www.impactjournals.com/oncotarget Figure 5: Effect of bortezomib on sarcoma tumors in vivo. A. PDX models used in this study. B. Levels of MAP17 mRNA in each model. C. Response to bortezomib. The left graph shows the average ±SD of tumor growth. The right graph shows the survival of the mouse cohorts with sacrifice by humane endpoint when the tumor reaches 1000 mm3. The sarcomas were subcutaneously engrafted and grown until all of the tumors reached 20 mm3 in volume. The mice were then treated for 4 weeks (5 days/eek). The cohorts of mice were either treated with bortezomib (1.0 mg/kg body weight, in 0.9% NaCl) or saline serum (0.9% NaCl). The mice were monitored daily for signs of distress and were weighed twice a week. The tumor size was measured using a caliper according to the following equation: tumor volume = [length x width2]/2. The experiments were terminated when the tumor reached 1000 mm3. Oncotarget67041 www.impactjournals.com/oncotarget and autophagy (Figure 6B, Supplementary Figure S4A). Moreover, we observed that MAP17 prevents the cytoprotective activation of NFκB and autophagy induced by bortezomib (Figure 6C, Supplementary Figure S4B) in sarcoma cell lines, a finding that has been reported previously for breast tumor cells [29]. Therefore, the inhibition of these protective pathways induced by MAP17 may explain the increase in sensitivity to bortezomib found in vivo. DISCUSSION High MAP17 levels correlate with a higher grade and poorer differentiation of tumors; therefore, therapies that can counteract MAP17 expression may lead to the promising application of known treatments to tumors with a poor prognosis. Sarcoma tumors that express MAP17 have a poor prognosis independent of the tumor origin. However, sarcoma cell lines and PDX tumors in vivo with high levels of MAP17 respond to the proteasome inhibitor bortezomib (Velcade, PS-341), approved by the FDA for the treatment of mantle cell lymphoma and multiple myeloma [33, 34]. Thus, our work provides first evidence that certain patients may benefit from this therapy that could be newly applied to second- or third-line sarcoma patients, currently without any other therapeutic option. MAP17 maintains a functional relationship with the proteasomal degradation pathway [29]. Used as a “seed”, MAP17 was found to be linked to proteins related to degradation pathways, mainly ubiquitination- and sumoylation-related pathways. We found that multiple myeloma patients with higher MAP17 mRNA levels Figure 6: Levels of NFκB and autophagy in sarcoma tumors or cell lines expressing MAP17. A. Western blots showing the levels of different basal protein levels in sarcoma PDX models. NFκB-p65 phosphorylated at Ser536, total NFκB-p65, LC3B and p62. Treatment with bortezomib was performed for 24 hrs. B. Quantification of Figure 6A, cell line AA. C. Quantification of Figure 6A, cell line AW. D. Western blots showing the levels of different proteins in AA and AW sarcoma cell lines overexpressing MAP17 or empty vector only. NFκB-p65 phosphorylated at Ser536, total NFκB-p65, LC3B and p62. Quantification is shown in Figure 4A. E. Western blots showing the levels of different proteins in AA and AW sarcoma cell lines overexpressing MAP17 or empty vector only, in response to bortezomib (+) or to solvent only (-). NFκB-p65 phosphorylated at Ser536, LC3B and p62 levels are shown. Quantification is shown in Figure 4B.