Predicting Ovarian Cancer Patients’ Clinical Response to Platinum-Based Chemotherapy by Their Tumor Proteomic Signatures

Ovarian cancer is the deadliest gynecologic malignancy in the United States with most patients diagnosed in the advanced stage of the disease. Platinum-based antineoplastic therapeutics is indispensable to treating advanced ovarian serous carcinoma. However, patients have heterogeneous responses to platinum drugs, and it is difficult to predict these interindividual differences before administering medication. In this study, we investigated the tumor proteomic profiles and clinical characteristics of 130 ovarian serous carcinoma patients analyzed by the Clinical Proteomic Tumor Analysis Consortium (CPTAC), predicted the platinum drug response using supervised machine learning methods, and evaluated our prediction models through leave-one-out cross-validation. Our data-driven feature selection approach indicated that tumor proteomics profiles contain information for predicting binarized platinum response (<i>P</i> < 0.0001). We further built a least absolute shrinkage and selection operator (LASSO)-Cox proportional hazards model that stratified patients into early relapse and late relapse groups (<i>P</i> = 0.00013). The top proteomic features indicative of platinum response were involved in ATP synthesis pathways and Ran GTPase binding. Overall, we demonstrated that proteomic profiles of ovarian serous carcinoma patients predicted platinum drug responses as well as provided insights into the biological processes influencing the efficacy of platinum-based therapeutics. Our analytical approach is also extensible to predicting response to other antineoplastic agents or treatment modalities for both ovarian and other cancers

Cell and patient data sets used for COXEN Predictor Training and Testing.

<p>NCI-60 and Peter-18 cell-line data sets were used to discover chemosensitivity biomarkers and to train multivariate statistical prediction models for paclitaxel and carboplatin, respectively. Bonome-185 set was used to select the biomarkers with the consistent directions of differential expression. Dressman-119 set was used to independently evaluate the trained predictors and to derive the optimal cutoff value of each predictor. UVA-55 set was purely used to test the predictability of the COXEN predictors in a prospective manner.</p

Prediction performance of COXEN predictors.

<p>The overall predictability (AUC) of identical COXEN predictors are summarized on the Dressman-119 and UVA-55 cohorts by AUC values with their 95% CIs and p-values. Cutoff values of COXEN predictors were derived by maximizing NPVs on the Dressman-119 cohort. Sensitivity, specificity, PPV, and NPV values were evaluated on both Dresseman-119 and independent UVA-55 cohort.</p

Clinical Characteristics of the UVA-55 Cohort.

<p>CR = Complete Response, PR = Partial Response, PD = Progressive Disease, PFS = Progression Free Survival, OS = Overall Survival, CI = Confidence Interval.</p

COXEN Biomarkers and Gene Networks for Carboplatin.

<p>Clustering heatmap analysis with major gene networks with x-axis responder (red) and non-responder (green) patients and y-axis Immunological disease/cell death entwork (red), Cell cycle/Connective tissue disorders/Inflammator disease network (green), Cellular movement/Hematological system/Immune cell trafficking network (yellow), and Free radical scavenging/cellular movement/cancer/cellular growth and proliferation network (blue).</p

Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion

<div><p>The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1β, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular “ectodomain” of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16^c354). Under a CMV, early enhancer plus chicken β actin promoter (CAG) MUC16^c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16^c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16^c354:p53^+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53^+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.</p></div

MUC16^c354 transgenic mice.

<p>A) Strategy for MUC16^c354 conditional construct. A CMV early enhancer plus the chicken β actin promoter (CAG) was used to drive the transcription of hrGFP between two loxPs and the downstream MUC16^c354 sequence. B) Southern blot shows 12 candidates of MUC16^c354 positive founders among 99 animals after the microinjection procedure. C) Western blot with anti-MUC16^c114 4H11 was used to identify founders 9 (~50 copies) and 36 (~10 copies) for MUC16^c354 mouse colony development. A5 is a positive control from a stable transfected SKOV3 with MUC16^c354. D) Histological analyses of tumors from double MUC16^c354:p53^+/- transgenic mice. Multiple sarcomas and lymphomas were identified in the double MUC16^c354:p53^+/- transgenic mice. Sections were stained with hematoxylin and eosin (H&E). Tumors included histocytic sarcoma in the uterus (I, Scale bar:100μm), liver (II, Scale bar:50μm), ovary (III, Scale bar:50μm) and bone marrow (IV, Scale bar:50μm); lymphoma in the ovary (V, Scale bar:50μm), kidney (VI, Scale bar:50μm), and lung (VII, Scale bar:50μm); and carcinoma in the lung (VIII, Scale bar:50μm). E) Transgenic mouse cancer-specific Kaplan-Meier survival curves: the MUC16^c354 mice (black line) showed no spontaneous tumor development over the first 18 months, similar to the wild type (WT, red dashed line). However, when MUC16^c354 mice were crossed with p53^+/- mice, the double transgenic MUC16^c354:p53^+/- mice (green dashed line) showed a significantly worse overall survival due to spontaneous tumor development compared to either the p53^+/- mice (red line) (p<0.014) or the MUC16^c354 mice. The number of tumors were p53^+/- mice 20/107; MUC16c354 and p53^+/- Mice 34/91; MUC16c354 1/72; and wild type 0/91.</p

Effect of N-glycosylation on MUC16 transformation.

<p>A) Matrigel invasion assay for 3T3 transfected cell lines, phrGFP control vector or MUC16^c114 or MUC16^3(N—A)c114 and MUC16^c114 treated with 0.1 μg/mL Tunicamycin. As seen earlier, MUC16^c114 cell lines were significantly different (p<0.0001) than the phrGFP vector control. The MUC16^3(N—A)c114 cell line was still significantly more invasive (** p = 0.007) compared to the phrGFP vector control. Treatment with the N-glycosylation inhibitor Tunicamycin significantly inhibited matrigel invasion compared to the untreated MUC16^c114 (##p = 0.0003), and MUC16^3(N—A)c114 is highly significant (### = p<0.0001) compared to MUC16^c114, suggesting that N-glycosylation is critical for MUC16-induced matrigel invasion. B) Matrigel invasion assay for 3T3 transfected cell lines compared to the phrGFP control vector. 3T3 cells transfected with MUC16^c114 were treated with media alone, with 5 μg/mL of control pFUSE hIgG1-Fc2 fusion protein, with 5 μg/mL of MUC16^c57-114-pFUSE hIgG1-Fc2 fusion protein, or with 5 μg/mL of ^117-244LGALS3-pFUSE hIgG1-Fc2 fusion protein, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126633#pone.0126633.s006" target="_blank">S4 Fig</a> As seen earlier, the MUC16^c114 cell line was much more invasive than the phrGFP vector control 3T3 cells (p<0.0001), and this increase in invasion was unaffected by exposure to pFUSE vector only protein. In contrast, MUC16^c114 cell line treated with MUC16^c57-114-pFUSE hIgG1-Fc2 fusion protein or ^117-244LGALS3-pFUSE hIgG1-Fc2 fusion protein demonstrated significant (# p = 0.0001) inhibition of matrigel invasion compared to MUC16^c114 control cells. C) Effect of MUC16 expression on ERK/AKT signaling. Phosphorylation of ERK1/2 (pT202/Y204) and AKT (S473) was increased in the 3T3 transfected with MUC16^c114; however, the effect was much diminished in 3T3 cells transfected with the MUC16^3(N—A)c114 vector. Despite the three asparagine—> alanine mutations, Western blot with the anti-MUC16 antibody, 4H11 mAb, showed a higher signal than either the phrGFP vector control or the native MUC16^c114 transfected cells, indicating that the high levels of MUC16^3(N—A)c114 protein is expressed in the transfected 3T3 cells, and surface expression was confirmed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126633#pone.0126633.s004" target="_blank">S2 Fig</a> β-Actin normalized densitometry quantification values are shown below each Western blot in the figure. D) MUC16-positive tumor growth in athymic nude mice. Two million tumor cells were introduced into the flank of 20 nu/nu mice, and the mice were observed for tumor formation. Tumors were measured by calipers twice weekly. The differences in mean tumor volume were significantly greater for mice bearing MUC16^c114 tumors (p<0.0001). As seen earlier, 3T3 MUC16^c114 transfectant was highly significant at *** p<0.0001 compared to the phrGFP control vector. However, MUC16^3(N—A)c1143T3 transfectants did not show any significance over phrGFP vector control 3T3 cells, indicating that the mutations of N-glycosylation dramatically decreased <i>in vivo</i> tumor growth and invasion.</p

Effects of truncated MUC16^c114 variants.

<p>A) Soft agar growth. 3T3 transfectants expressing either internal or external domain portions of MUC16^c114 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126633#pone.0126633.s003" target="_blank">S1 Fig</a>) were layered on soft agar, as described in the Material and Methods section. Colonies were counted and plotted. The data shown represent one of three experiments. Soft agar growth rates for MUC16^c80 and MUC16^c86 were significant (# p = 0.0111 and ## p = 0.0258, respectively) compared to MUC16^c114, whereas a higher level of significance (###p<0.0001) was seen with MUC16^c80 transfectant compared to MUC16^c86. B) Matrigel invasion assay for 3T3 cell lines transfected with either phrGFP control vector or with MUC16 carboxy-terminal constructs. Each assay was performed two or more times in triplicate and counted by hand. MUC16^c80 transfectant was significant (# p = 0.0172) compared to the MUC16^c114 cell line. C) Effect of MUC16 expression on ERK/AKT signaling. Transfected 3T3 cells were examined for activation of the ERK/AKT signaling pathways. Phosphorylation of ERK1/2 (pT202/Y204) and AKT (S473) was increased following MUC16^c114 transfection; however, the signals were lower with either the MUC16^c80 or MUC16^c86 constructs. β-Actin normalized densitometry quantification values are shown below each Western blot. D) MUC16-positive tumor growth in athymic nude mice. Two million tumor cells were introduced into the flank of 20 nu/nu mice, and the mice were observed for tumor formation. Tumors were measured by calipers twice weekly. The differences in mean tumor volume were significantly greater for mice bearing MUC16 ectodomain positive tumors. 3T3 MUC16^c114 and 3T3 MUC16^c86 transfectants were significantly different compared to MUC16^c80 (### p<0.0001) and vector only animals.</p

Effect of MUC16 in 3T3 cells.

<p>A) Soft agar growth of 3T3 transfectants in 60 mm dishes. After 14 days, colonies were counted and plotted. The data shown in the table represent one of three similar experiments (*** p < 0.0001) compared to the phrGFP control vector; B) Matrigel invasion assay for 3T3 cell lines following stable transfections with either the phrGFP control vector, MUC16^c114-phrGFP or MUC16^c344 phrGFP carboxy-terminal constructs. Each assay was performed two or more times in triplicate and counted by hand. Both MUC16^c114 3T3 and MUC16^c344 3T3 cell lines were significantly more invasive (*** p<0.0001) compared to the phrGFP 3T3 vector control, and the MUC16^c344 cell line is significantly more invasive than the MUC16^c114 cell line (# p = 0.0354). C) Expression of metastasis and invasion genes induced by MUC16^c114 and MUC16^c344 expression. A SuperArray panel of 80 invasion/metastasis gene transcripts was examined for MUC16-positive and vector only cell lines. The expression of selected chemotactic, adhesion, and invasion transcripts was measured in 3T3 MUC16^c114 or 3T3-MUC16^c344 cell lines (each of three triplicates was examined in duplicate and compared to the phr vector only controls by chi square testing). The p value for each transcript, adjusted for repeated measures, is shown in the table. All genes with changes at the corrected p<0.05 or below level are included. D) Transfected 3T3 cells were examined for activation of the ERK/AKT signaling pathways compared to the vector only controls. Phosphorylation of ERK1/2 (pT202/Y204) and AKT (S473) was increased following MUC16^c114 and MUC16^c344 constructs, compared to the phrGFP vector. Activation of both pathways was seen in each of the cell lines. β-Actin normalized densitometry quantification values are shown below each Western blot band. E) MUC16 transfectant tumor growth in athymic nude mice. Two million tumor cells were introduced into the flank of 15 nu/nu mice, and the mice were observed for tumor formation. Tumors were measured by calipers twice weekly. The differences in mean tumor volume were significantly greater for mice bearing MUC16-positive tumors (both lines p<0.0001 compared to the phrGFP control vector).</p